Methods for aiding in diagnosing and evaluating a mild traumatic brain injury in a human subject using cardiac troponin I

ABSTRACT

Disclosed herein are methods that aid in the diagnosis and evaluation of a human subject that has sustained or may have sustained an injury to the head, such as mild or moderate, severe, or moderate to severe traumatic brain injury (TBI), using cTnI. Also disclosed are methods for determining whether to perform a head computerized tomography on a subject by detecting levels of cTnI. Finally, also disclosed are methods of outcome in subjects suffering from a mild TBI.

RELATED APPLICATION INFORMATION

This application claims priority to U.S. Application No. 62/512,688filed on May 30, 2017, U.S. Application No. 62/512,710 filed on May 30,2017 and U.S. Application No. 62/528,214 filed on Jul. 3, 2017, thecontents of each of which are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates to methods for aiding in diagnosing andevaluating a human subject that has sustained or may have sustained an(or has an has an actual or suspected) injury to the head, such as mildtraumatic brain injury (TBI), by detecting levels of cardiac troponin I(cTnI) in samples (or biological samples) taken from the human subjectat time points of injury after the subject has sustained or may havesustained (or has an has an actual or suspected) the injury to the head.The present disclosure also provides methods of determining whether toperform a head computerized tomography on a subject based on detectingvarious levels of cTnI. The present disclosure also provides methods ofpredicting outcome in subjects suffering from a TBI.

BACKGROUND

More than 5 million mild traumatic brain injuries (TBIs) occur each yearin the United States alone. Currently, there is no simple, objective,accurate measurement available to help in patient assessment. In fact,much of TBI evaluation and diagnosis is based on subjective data.Unfortunately, objective measurements such as head CT and Glasgow ComaScore (GCS) are not very comprehensive or sensitive in evaluating mildTBI. Moreover, head CT is unrevealing for the vast majority of the timefor mild TBI, is expensive, and exposes the patient to unnecessaryradiation. Additionally, a negative head CT does not mean the patienthas been cleared from having a concussion; rather it just means certaininterventions, such as surgery, are not warranted. Clinicians andpatients need objective, reliable information to accurately evaluatethis condition to promote appropriate triage and recovery. To date,limited data have been available for the use of cardiac troponin I inthe acute care setting or hyperacute care setting (very early acute timepoints after injury) to aid in patient evaluation and management.

Mild TBI or concussion is much harder to objectively detect and presentsan everyday challenge in emergency care units globally. Concussionusually causes no gross pathology, such as hemorrhage, and noabnormalities on conventional computed tomography scans of the brain,but rather rapid-onset neuronal dysfunction that resolves in aspontaneous manner over a few days to a few weeks. Approximately 15% ofmild TBI patients suffer persisting cognitive dysfunction. There is anunmet need for mild TBI victims on scene, in emergency rooms andclinics, in the sports area and in military activity (e.g., combat).

Current algorithms for assessment of the severity of brain injuryinclude Glasgow Coma Scale score and other measures. These measures mayat times be adequate for relating acute severity but are insufficientlysensitive for subtle pathology which can result in persistent deficit.GCS and other measures also do not enable differentiation among types ofinjury and may not be adequate. Thus patients grouped into a single GCSlevel entering a clinical trial may have vastly heterogeneous severityand type of injury. Because outcomes also vary accordingly,inappropriate classification undermines the integrity of a clinicaltrial. Improved classification of injury will enable more precisedelineation of disease severity and type for TBI patients in clinicaltrials.

Additionally, current brain injury trials rely on outcome measures suchas Glasgow Outcome Scale Extended, which capture global phenomena butfail to assess for subtle differences in outcome. Thus 30 consecutivetrials for brain injury therapeutics have failed. Sensitive outcomemeasures are needed to determine how well patients have recovered frombrain injury in order to test therapeutics and prophylactics.

Traumatic brain injury (TBI) patients are at least three times morelikely to die from cardiovascular causes than the general population.Cardiac injury from TBI is also associated with neurogenic pulmonaryedema. The phenomenon of cardiac injury in neurologic conditions hasbeen described in patients with spontaneous subarachnoid hemorrhage andis believed to result from a fulminant surge in catecholamine levels.However, the mechanisms underlining excess cardiovascular mortality inTBI have been poorly studied and therefore are not well understood.Consequently, it is unclear whether: the onset of cardiac injury occursin the acute or chronic phase of TBI; there are particular sub-types ofTBI that are preferentially affected by cardiac injury; and what thebiological triggers of cardiac injury in TBI are. A number ofretrospective studies have investigated myocardial injury in the acutephase of TBI. Using conventional cardiac troponin assays, these studieshave reported cardiac injury (as determined by elevated troponin levels)within 24 hours of injury in 30% of severe TBI patient. Cardiac injuryin TBI is associated with injury severity and age. TBI patients withcardiac injury have a higher risk of in-patient mortality than thosewithout cardiac injury. However, these findings are subject to spectrumbias since they are derived from retrospective studies and troponinmeasurements were performed at the discretion of clinicians (they arerarely done in the routine care of TBI patients). Furthermore, theassociation between cardiac injury and neurologic outcome in TBI has notbeen studied. Additionally, the role of cardiac injury in mild andmoderate TBI has not been studied.

SUMMARY

In some embodiments, the present disclosure relates to a method foraiding in the diagnosis and evaluation of mild traumatic brain injury ina human subject. The method can comprise the steps of:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or or suspected injury to the head tomeasure or detect a level of cardiac troponin I (cTnI); and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI or (2) a mild traumatic braininjury when the level of cTnI in the sample is lower than a referencelevel of cTnI.

In some embodiments of the above method, the subject is diagnosed ordetermined to have sustained a mild traumatic brain injury. In otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a moderate traumatic brain injury. In yet otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a severe traumatic brain injury. In yet otherembodiments, the subject is diagnosed or determined to have sustained amoderate to severe traumatic brain injury.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments in the above method, the subject is suspected ashaving moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

In some embodiments in the above method, the reference level iscorrelated with (corresponds to) subjects having a moderate, severe, ora moderate to severe traumatic brain injury. In some embodiments in theabove method, the reference level is correlated with (corresponds to) amoderate traumatic brain injury. In other embodiments of the abovemethod, the reference level is correlated with (corresponds to) a severetraumatic brain injury.

In some embodiments, the subject may be suspected as having mild TBIbased on the Glasgow Coma Scale score. In other aspects, the subject maybe suspected of having a moderate TBI based on the Glasgow Coma Scalescore. In other aspects, the subject may be suspected of having a severeTBI based on the Glasgow Coma Scale Score. In other aspects, the subjectmay be suspect as having a moderate to severe TBI based on the GlasgowComa scale score. In other aspects, the reference level of GFAP or thereference level correlates with or correspond to a Glasgow Coma Scalescore of 13-15 (a mild TBI). In other aspects, the reference levelcorrelates or correspond to a Glasgow Coma Scale score of 3-8 (a severeTBI). In other aspects, the reference level correlates or correspond toa Glasgow Coma Scale score of 9-13 (a moderate TBI). In other aspects,the reference level correlates with or correspond to a Glasgow ComaScale score of 3-12 (a moderate to severe TBI).

In some embodiments in the above method, the reference level for cTnI isabout 1.94 pg/mL, about 2.54 pg/mL, about 21.23 pg/mL, or about 43.79pg/mL. In some embodiments in the above method, the reference level forcTnI is about 1.94 pg/mL. In some embodiments in the above method, thereference level for cTnI is about 2.54 pg/mL. In some embodiments in theabove method, the reference level for cTnI is about 21.23 pg/mL. In someembodiments in the above method, the reference level for cTnI is about43.79 pg/mL.

In some embodiments in the above method, the reference level is (a)determined by an assay having a sensitivity of between at least about85% to 100% and a specificity of between at least about 30% to 100%; (b)determined by an assay having a sensitivity of at least about 87.5% anda specificity of at least about 31%; or (c) between at least about 1pg/mL to about 50 pg/mL. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%. In someembodiments in the above method, the reference level is between at leastabout 1 pg/mL to about 50 pg/mL.

In some embodiments in the above method, the sample is taken withinabout 30 minutes, within about 1 hour, within about 2 hours, withinabout 3 hours, within about 4 hours, within about 5 hours, within about6 hours, within about 7 hours, within about 8 hours, within about 9hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of actual or suspected injury to the head. Specifically, in someembodiments of the above method, the sample is taken within about 30minutes of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 1 hour of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 2 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 3 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 4 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 5hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 6 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 7 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 8 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about9 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 10 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 11 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 12 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 13 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about14 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 15 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 16 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 17 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 18 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about19 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 20 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 21 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 22 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 23 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about24 hours of actual or suspected injury to the head.

In some embodiments, the above method further comprises treating thesubject assessed as having a moderate, severe or moderate to severetraumatic brain injury with a traumatic brain injury treatment. In someembodiments, the above method further comprises monitoring the subjectassessed as having a moderate, severe, or moderate to severe traumaticbrain injury before treatment with a traumatic brain injury treatment.In some embodiments, the above method further comprises monitoring thesubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury after treatment with a traumatic brain injurytreatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In another embodiment, the present disclosure relates to aiding in thedetermination of whether to perform a head computerized tomography (CT)scan on a human subject that has sustained or may have sustained an (orhas an an actual or suspected) injury to the head. The method cancomprise the steps of:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or suspected injury to the head tomeasure or detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

In some embodiments of the above method, a CT scan is performed on thesubject. In other embodiments of the above method, a CT scan is notperformed on the subject.

In some embodiments in the above method, the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of an actual or suspected injury to the head.Specifically, in some embodiments of the above method, the sample istaken within about 30 minutes of an actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 1 hour of an actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about2 hours of an actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 3hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 4 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 5 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 6 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about7 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 8 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 9 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 10 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 11 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about12 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 13 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 14 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 15 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 16 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about17 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 18 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 19 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 20 hours of actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 21 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about22 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 23 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 24 hours of actual or suspectedinjury to the head.

In some embodiments of the above-described method, the subject hasreceived a CT scan before or after the assay is performed, and whereinthe subject is suspected as having a TBI based on the CT scan result. Insome embodiments, the subject may be suspected of having a traumaticbrain injury based on a CT scan that already was performed. For example,depending upon a subject's medical condition (such as, if the patient isunconscious), a CT scan may be conducted shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a CT scan maybe performed prior to the assay being performed to confirm and determinewhether the subject has a mild or a moderate, severe, or a moderate tosevere TBI. After the assay is performed, one or more subsequent CTscans can be performed based on the results of the assay as part of thephysician's (or other medical personnel's) management of the TBI (suchas, for example, to determine whether surgical and/or pharmacologicalintervention may be required). In other embodiments, the subject may nothave received a CT scan before the assay is performed.

In some embodiments in the above method, the subject is suspected ofhaving a traumatic brain injury based on the CT scan. In someembodiments, the subject is diagnosed as having a traumatic brain injurybased on the CT scan. In other embodiments, the subject is diagnosed asnot having a traumatic brain injury based on the CT scan.

In some embodiments in the above method, the reference level iscorrelated with (corresponds to) a positive head computed tomography.

In some embodiments in the above method, the reference level iscorrelated with (corresponds to) control subjects that have notsustained a head injury.

In some embodiments in the above method, the reference level for cTnI isabout 1.65 pg/mL, about 2.16 pg/mL, about 14.75 pg/mL, or about 30.43pg/mL. In some embodiments in the above method, the reference level forcTnI is about 1.65 pg/mL. In some embodiments in the above method, thereference level for cTnI is about 2.16 pg/mL. In some embodiments in theabove method, the reference level for cTnI is about 14.75 pg/mL. In someembodiments in the above method, the reference level for cTnI is about30.43 pg/mL.

In some embodiments in the above method, the reference level is (a)determined by an assay having a sensitivity of between at least about65% to 100% and a specificity of between at least about 30% to 100%; (b)determined by an assay having a sensitivity of at least about 85% and aspecificity of at least about 33%; or (c) between at least about 1.0pg/mL to about 50 pg/mL. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%. In someembodiments in the above method, the reference level is between at leastabout 1.0 pg/mL to about 50 pg/mL.

In another embodiment, the present disclosure relates to a method foraiding in the diagnosis and evaluation of mild traumatic brain injury ina human subject. The method can comprise the steps of:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 3 hours to about 6 hours after the firstsample, wherein the samples are biological samples;b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; andc) confirming the occurrence of a moderate, severe or moderate to severetraumatic brain injury if the level of cTnI detected has increased fromthe first sample to the second sample and confirming the absence of mildtraumatic brain injury if the level of cTnI detected has remainedunchanged or has decreased from the first sample to the second sample.

In some embodiments of the above method, the subject is confirmed tohave a mild traumatic brain injury. In other embodiments of the abovemethod, the subject is confirmed to have a moderate traumatic braininjury. In yet other embodiments of the above method, the subject isconfirmed to have to have a severe traumatic brain injury. In yet otherembodiments, the subject is confirmed to have to have a moderate tosevere traumatic brain injury.

In some embodiments in the above method, the first sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of an actual or suspected injury to the head.Specifically, in some embodiments of the above method, the sample istaken within about 30 minutes of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 1 hour of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 2hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 3 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 4 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 5 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about6 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 7 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 8 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 9 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 10 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 11hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 12 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 13 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 14 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 15 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 16hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 17 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 18 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 19 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 20 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 21hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 22 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 23 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 24 hours of actual or suspected injury to the head.

In some embodiments in the above method, the subject has an abnormalhead CT.

In some embodiments in the above method, the amount of cTnI in the firstsample is from about 1.0 to about 50 pg/mL.

In some embodiments in the above method, the amount of cTnI in thesecond sample is from about 1.0 to about 50 pg/mL.

In some embodiments in the above method, the method further comprisestreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment. In some embodiments, the above method further comprisesmonitoring the subject assessed as having a moderate, severe, ormoderate to severe traumatic brain injury before treatment with atraumatic brain injury treatment. In some embodiments, the above methodfurther comprises monitoring the subject assessed as having a moderate,severe, or moderate to severe traumatic brain injury after treatmentwith a traumatic brain injury treatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In another embodiment, the present disclosure relates to a method foraiding in the diagnosis and evaluation of mild traumatic brain injury ina human subject. The method can comprise the steps of:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 1 hours to about 4 hours after the firstsample, wherein the samples are biological samples;b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; andc) confirming the occurrence of a moderate, severe or moderate to severetraumatic brain injury if the level of cTnI detected has increased fromthe first sample to the second sample and confirming the absence of mildtraumatic brain injury if the level of cTnI detected has remainedunchanged or has decreased from the first sample to the second sample.

In some embodiments of the above method, the subject is confirmed tohave a mild traumatic brain injury. In other embodiments of the abovemethod, the subject is confirmed to have a moderate traumatic braininjury. In yet other embodiments of the above method, the subject isconfirmed to have to have a severe traumatic brain injury. In yet otherembodiments, the subject is confirmed to have to have a moderate tosevere traumatic brain injury.

In some embodiments in the above method, the first sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of an actual or suspected injury to the head.Specifically, in some embodiments of the above method, the sample istaken within about 30 minutes of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 1 hour of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 2hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 3 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 4 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 5 hours of actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about6 hours of actual or suspected injury to the head. In other embodimentsof the above method, the sample is taken within about 7 hours of actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 8 hours of actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 9 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 10 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 11hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 12 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 13 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 14 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 15 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 16hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 17 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 18 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 19 hours of actual or suspected injury to the head.In other embodiments of the above method, the sample is taken withinabout 20 hours of actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 21hours of actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 22 hours of actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 23 hours of actual or suspected injuryto the head. In other embodiments of the above method, the sample istaken within about 24 hours of actual or suspected injury to the head.

In some embodiments in the above method, the subject has an abnormalhead CT.

In some embodiments in the above method, the amount of cTnI in the firstsample is from about 1.0 to about 50 pg/mL.

In some embodiments in the above method, the amount of cTnI in thesecond sample is from about 1.0 to about 50 pg/mL.

In some embodiments in the above method, the method further comprisestreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment. In some embodiments, the above method further comprisesmonitoring the subject assessed as having a moderate, severe, ormoderate to severe traumatic brain injury before treatment with atraumatic brain injury treatment. In some embodiments, the above methodfurther comprises monitoring the subject assessed as having a moderate,severe, or moderate to severe traumatic brain injury after treatmentwith a traumatic brain injury treatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In another embodiment, the present disclosure relates to a method ofaiding in the diagnosis and evaluation of a human subject that hassustained or may have sustained an (or has an actual or suspected)injury to the head. The method can comprise the steps of:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an injury or suspected injury to the head to measureor detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI or (2) a mild traumatic braininjury when the level of cTnI in the sample is lower than a referencelevel of cTnI.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments, the subject may be suspected as having mild TBIbased on the Glasgow Coma Scale score. In other aspects, the subject maybe suspected of having a moderate TBI based on the Glasgow Coma Scalescore. In other aspects, the subject may be suspected of having a severeTBI based on the Glasgow Coma Scale Score. In other aspects, the subjectmay be suspect as having a moderate to severe TBI based on the GlasgowComa scale score. In other aspects, the reference level of GFAP or thereference level correlates with or correspond to a Glasgow Coma Scalescore of 13-15 (a mild TBI). In other aspects, the reference levelcorrelates or correspond to a Glasgow Coma Scale score of 3-8 (a severeTBI). In other aspects, the reference level correlates or correspond toa Glasgow Coma Scale score of 9-13 (a moderate TBI). In other aspects,the reference level correlates with or correspond to a Glasgow ComaScale score of 3-12 (a moderate to severe TBI).

In some embodiments in the above method, the reference level for cTnI isabout 1.15 pg/mL. In some embodiments in the above method, the referencelevel for cTnI is about 1.29 pg/mL.

In some embodiments in the above method, the reference level is (a)determined by an assay having a sensitivity of between at least about85% to 100% and a specificity of between at least about 30% to 100%; (b)determined by an assay having a sensitivity of at least about 87.5% anda specificity of at least about 31%; or (c) between at least about 0.5pg/mL to about 30 pg/mL. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%. In someembodiments in the above method, the reference level is between at leastabout 0.5 pg/mL to about 30 pg/mL.

In some embodiments in the above method, the sample is taken withinabout 5 minutes, within about 10 minutes, within about 12 minutes,within about 15 minutes, within about 20 minutes, within about 30minutes, within about 60 minutes, or within about 90 minutes after anactual or suspected injury to the head. In some embodiments of theabove-identified method, the sample is taken within about 5 minutesafter an actual or suspected injury to the head. In other embodiments,the sample is taken within about 10 minutes of suspected injury to thehead. In yet other embodiments, the sample is taken within about 12minutes of suspected injury to the head. In yet other embodiments, thesample is taken within about 15 minutes of suspected injury to the head.In yet other embodiments, the sample is taken within about 20 minutes ofsuspected injury to the head. In yet other embodiments, the sample istaken within about 60 minutes of suspected injury to the head. In yetsill other embodiments, the sample is taken within about 90 minutes ofsuspected injury to the head.

In some embodiments, the above method further comprises monitoring thesubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury before treatment with a traumatic brain injurytreatment. In some embodiments, the above method further comprisesmonitoring the subject assessed as having a moderate, severe, ormoderate to severe traumatic brain injury after treatment with atraumatic brain injury treatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In another embodiment, the present disclosure relates to a method ofaiding in the determination of whether to perform a head computerizedtomography (CT) scan on a human subject that has sustained or may havesustained an (or has an actual or suspected) injury to the head. Themethod can comprise the steps of:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

In some embodiments of the above method, a CT scan is performed on thesubject. In other embodiments of the above method, a CT scan is notperformed on the subject.

In some embodiments of the above-described method, the subject hasreceived a CT scan before or after the assay is performed, and whereinthe subject is suspected as having a TBI based on the CT scan result. Insome embodiments, the subject may be suspected of having a traumaticbrain injury based on a CT scan that already was performed. For example,depending upon a subject's medical condition (such as, if the patient isunconscious), a CT scan may be conducted shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a CT scan maybe performed prior to the assay being performed to confirm and determinewhether or not the subject has a mild or moderate, severe or a moderateto severe TBI. After the assay is performed, one or more subsequent CTscans can be performed based on the results of the assay as part of thephysician's (or other medical personnel's) management of the TBI (suchas, for example, to determine whether surgical and/or pharmacologicalintervention may be required). In other embodiments, the subject may nothave received a CT scan before the assay is performed.

In some embodiments in the above method, the subject is suspected ofhaving a traumatic brain injury based on the CT scan. In someembodiments, the subject is diagnosed as having a traumatic brain injurybased on the CT scan. In some embodiments, the subject is diagnosed asnot having a traumatic brain injury based on the CT scan.

In some embodiments in the above method, the reference level iscorrelated with (or corresponds to) a positive head computed tomography.

In some embodiments in the above method, the reference level iscorrelated with (or corresponds with) control subjects that have notsustained a head injury.

In some embodiments in the above method, the reference level for cTnI isabout 5.8 pg/mL. In some embodiments in the above method, the referencelevel for cTnI is about 4.7 pg/mL.

In some embodiments in the above method, the reference level is (a)determined by an assay having a sensitivity of between at least about65% to 100% and a specificity of between at least about 30% to 100%; (b)determined by an assay having a sensitivity of at least about 85% and aspecificity of at least about 33%; or (c) between at least about 0.5pg/mL to about 25 pg/mL. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%. In some embodiments in the above method, thereference level is determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%. In someembodiments in the above method, the reference level is between at leastabout 0.5 pg/mL to about 25 pg/mL.

In some embodiments in the above method, the sample is taken withinabout 5 minutes, within about 10 minutes, within about 12 minutes,within about 15 minutes, within about 20 minutes, within about 30minutes, within about 60 minutes, or within about 90 minutes after anactual or suspected injury to the head. In some embodiments of theabove-identified method, the sample is taken within about 5 minutesafter an actual or suspected injury to the head. In other embodiments,the sample is taken within about 10 minutes of an actual or suspectedinjury to the head. In yet other embodiments, the sample is taken withinabout 12 minutes of an actual or suspected injury to the head. In yetother embodiments, the sample is taken within about 15 minutes of anactual or suspected injury to the head. In yet other embodiments, thesample is taken within about 20 minutes of an actual suspected injury tothe head. In yet other embodiments, the sample is taken within about 60minutes of an actual or suspected injury to the head. In yet sill otherembodiments, the sample is taken within about 90 minutes of an actual orsuspected injury to the head.

In yet another embodiment, the present disclosure relates to methods oftreating a mild or moderate, severe, or moderate to severe TBI, themethod comprising: a) performing an assay on a sample obtained from thesubject within about 24 hours after an injury to the head to measure ordetect a level of cTnI; b) determining whether the subject has sustaineda mild or a moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI or (2) a mild traumatic braininjury when the level of cTnI in the sample is lower than a referencelevel of cTnI; and c) treating the subject assessed as having a mild,moderate, severe, or moderate to severe traumatic brain injury with atraumatic brain injury treatment.

Embodiments of the above method further involve monitoring the subjectassessed as having a mild traumatic brain injury. Embodiments of theabove method further involve monitoring the subject assessed as having amoderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above method, the traumatic brain injurytreatment for a subject suffering from a mild TBI can involve having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. In other embodiments of the above method,the traumatic brain injury treatment for a subject suffering from amoderate, severe, or moderate to severe TBI, the treatment involves theadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt or providing of oneor more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof. Optionally, such methods may alsoinvolve providing one or more cardioprotective therapies. Suchcardioprotective therapies can be administered in combination with thetreatments for the TBI or alone without any TBI treatment, depending onthe circumstances.

In yet another embodiment, the present disclosure relates to a method oftreating a mild or moderate, severe, or moderate to severe traumaticbrain injury in a human subject, the method comprising: a) performing anassay on samples from the human subject to measure or detect a level ofcTnI in a first sample and a second sample, wherein the first sample istaken from the human subject at a first time point and the second sampleis taken from the human subject about 3 hours to about 6 hours after thefirst sample, wherein the samples are biological samples; b) determiningwhether the amount of cTnI has increased or decreased from the firstsample to the second sample; c) confirming the occurrence of moderate tosevere traumatic brain injury if the level of cTnI detected hasincreased from the first sample to the second sample and confirming theabsence of mild traumatic brain injury if the level of cTnI detected hasremained unchanged or has decreased from the first sample to the secondsample; and d) treating the subject assessed as having a mild, moderate,severe, or moderate to severe traumatic brain injury with a traumaticbrain injury treatment.

Embodiments of the above method further involve monitoring the subjectassessed as having a mild traumatic brain injury. Embodiments of theabove method further involve monitoring the subject assessed as having amoderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above method, the traumatic brain injurytreatment for a subject suffering from a mild TBI can involve having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. In other embodiments of the above method,the traumatic brain injury treatment for a subject suffering from amoderate, severe, or moderate to severe TBI, the treatment involves theadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt or providing of oneor more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof. Optionally, such methods may alsoinvolve providing one or more cardioprotective therapies. Suchcardioprotective therapies can be administered in combination with thetreatments for the TBI or alone without any TBI treatment, depending onthe circumstances.

In yet another embodiment, the present disclosure relates to a method oftreating a mild or moderate, severe, or moderate to severe traumaticbrain injury in a human subject, the method comprising: a) performing anassay on samples from the human subject to measure or detect a level ofcTnI in a first sample and a second sample, wherein the first sample istaken from the human subject at a first time point and the second sampleis taken from the human subject about 1 hours to about 4 hours after thefirst sample, wherein the samples are biological samples; b) determiningwhether the amount of cTnI has increased or decreased from the firstsample to the second sample; c) confirming the occurrence of moderate tosevere traumatic brain injury if the level of cTnI detected hasincreased from the first sample to the second sample and confirming theabsence of mild traumatic brain injury if the level of cTnI detected hasremained unchanged or has decreased from the first sample to the secondsample; and d) treating the subject assessed as having a mild, moderate,severe, or moderate to severe traumatic brain injury with a traumaticbrain injury treatment.

Embodiments of the above method further involve monitoring the subjectassessed as having a mild traumatic brain injury. Embodiments of theabove method further involve monitoring the subject assessed as having amoderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above method, the traumatic brain injurytreatment for a subject suffering from a mild TBI can involve having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. In other embodiments of the above method,the traumatic brain injury treatment for a subject suffering from amoderate, severe, or moderate to severe TBI, the treatment involves theadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt or providing of oneor more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof. Optionally, such methods may alsoinvolve providing one or more cardioprotective therapies. Suchcardioprotective therapies can be administered in combination with thetreatments for the TBI or alone without any TBI treatment, depending onthe circumstances.

In still yet another embodiment, the present disclosure relates to amethod for aiding in predicting or predicting the outcome of humansubjects having mild traumatic brain injury, the method comprising: a)performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin 24 hours after a injury to the head and the second sample istaken from the human subject about 0 to about 4 hours after the firstsample, wherein the samples are biological samples; b) determiningwhether the amount of cTnI has increased or decreased from the firstsample to the second sample; and c) predicting an unfavorable outcome inthe human subject if the level of cTnI detected has increased from thefirst sample to the second sample by at least about 20% and predicting afavorable outcome in the human subject if the level of cTnI detected hasremained unchanged, decreased, or increased less than about 20% from thefirst sample to the second sample.

In some embodiments of the above described method, the first sample istaken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours after an injury to the head. In anotherembodiment, the first sample is taken from the subject within about 30minutes after an injury to the head. In another embodiment, the firstsample is taken from the subject within about 1 hour after an injury tothe head. In some embodiments of the above method, the first sample istaken from the subject within about 2 hours after an injury to the head.In some embodiments of the above method, the first sample is taken fromthe subject within about 3 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 4 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 5 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 6 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 7 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 8 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 9 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 10 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 11 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 12 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 13 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 14 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 15 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 16 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 17 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 18 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 19 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 20 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 21 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 22 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 23 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 24 hours after an injury to the head.

In some embodiments of the above method, the method predicts a favorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 6 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 6 months after an injury to the head.

In some embodiments of the above method, the subject has a normal headCT scan.

In some embodiments of the above method, the subject has an abnormalhead CT scan.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether or not the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments of the above method, the subject has an ExtendedGlasgow Outcome (GOSE) score of 5 or less. In some embodiments of theabove method, the GOSE score is obtained before or after the assay isperformed.

In some embodiments of the above method, the subject (having a mild TBIor a moderate, severe, or moderate to severe TBI) that is assessed ashaving an unfavorable outcome is treated with a traumatic brain injurytreatment. In some embodiments of the above method, the traumatic braininjury treatment for a subject suffering from a mild TBI can involvehaving the subject rest for a certain period of time, abstain fromphysical activities for a certain period of time, administration of oneor more therapeutics (e.g., drugs to provide relief for a headache ormigraine, etc.) or combinations thereof. In other embodiments of theabove method, the traumatic brain injury treatment for a subjectsuffering from a moderate, severe, or moderate to severe TBI, thetreatment involves the administration of one or more therapeutics (e.g.,drugs such as diuretics, anti-seizure drugs), performing one or moresurgical procedures (e.g., such as removal of a hematoma, repairing askull fracture, decompressive crainiectomy, etc.), receipt or providingof one or more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof.

In some embodiments of the above method, the subject having beenassessed as having an unfavorable outcome may also be monitored. Saidsubject may be monitored regardless of whether the subject is receivinga traumatic brain injury treatment or other treatment (such as one ormore cardioprotective therapies).

The present disclosure is directed to a method for aiding in predictingor predicting the outcome of human subjects having mild traumatic braininjury, the method comprising: a) performing an assay on a sampleobtained from the subject within about 28 hours after an injury to thehead to measure or detect a level of cTnI, wherein the sample is abiological sample; and

b) predicting an unfavorable outcome in the human subject if the levelof cTnI in the sample is higher than a reference level of cTnI andpredicting a favorable outcome in the human subject if the level of cTnIin the sample is lower than a reference level of cTnI.

In some embodiments of the above described method, the first sample istaken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of after an injury to the head. Inanother embodiment, the first sample is taken from the subject withinabout 30 minutes after an injury to the head. In another embodiment, thefirst sample is taken from the subject within about 1 hour after aninjury to the head. In some embodiments of the above method, the firstsample is taken from the subject within about 2 hours after an injury tothe head. In some embodiments of the above method, the first sample istaken from the subject within about 3 hours after an injury to the head.In some embodiments of the above method, the first sample is taken fromthe subject within about 4 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 5 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 6 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 7 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 8 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 9 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 10 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 11 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 12 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 13 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 14 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 15 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 16 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 17 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 18 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 19 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 20 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 21 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 22 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 23 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 24 hours after an injury to the head.

In some embodiments of the above method, the method predicts a favorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 6 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 6 months after an injury to the head.

In some embodiments of the above method, the subject has a normal headCT scan.

In some embodiments of the above method, the subject has an abnormalhead CT scan.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments of the above method, the subject has an ExtendedGlasgow Outcome (GOSE) score of 5 or less. In some embodiments of theabove method, the GOSE score is obtained before or after the assay isperformed.

In some embodiments of the above method, the reference level isreference level is determined by an assay having a sensitivity ofbetween at least about 80% to 100% and a specificity of between at leastabout 45% to 100%. In some embodiments of the above method, thereference level is reference level is determined by an assay having asensitivity of at least about 83.3% and a specificity of at least about54.9%. In some embodiments of the above method, the reference level isreference level is determined by an assay having a sensitivity of atleast about 100% and a specificity of at least about 49.2%. In someembodiments of the above method, the reference level is reference levelbetween at least about 1 pg/mL to about 50 pg/mL.

In other embodiments of the above method, the reference level for cTnIis about 5.6 pg/mL. In other embodiments of the above method, thereference level for cTnI is about 5.7 pg/mL.

In some embodiments of the above method, the subject (having a mild TBIor a moderate, severe, or moderate to severe TBI) that is assessed ashaving an unfavorable outcome is treated with a traumatic brain injurytreatment. In some embodiments of the above method, the traumatic braininjury treatment for a subject suffering from a mild TBI can involvehaving the subject rest for a certain period of time, abstain fromphysical activities for a certain period of time, administration of oneor more therapeutics (e.g., drugs to provide relief for a headache ormigraine, etc.) or combinations thereof. In other embodiments of theabove method, the traumatic brain injury treatment for a subjectsuffering from a moderate, severe, or moderate to severe TBI, thetreatment involves the administration of one or more therapeutics (e.g.,drugs such as diuretics, anti-seizure drugs), performing one or moresurgical procedures (e.g., such as removal of a hematoma, repairing askull fracture, decompressive crainiectomy, etc.), receipt or providingof one or more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof.

In some embodiments of the above method, the subject having beenassessed as having an unfavorable outcome may also be monitored. Saidsubject may be monitored regardless of whether the subject is receivinga traumatic brain injury treatment or other treatment (such as one ormore cardioprotective therapies).

In yet another embodiment, the present disclosure is directed to amethod for aiding in predicting or predicting the outcome of humansubjects having mild traumatic brain injury, the method comprising: a)performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 4 hours after the first sample, wherein thesamples are biological samples; b) determining whether the amount ofcTnI has increased or decreased from the first sample to the secondsample; and c) predicting an unfavorable outcome in the human subject ifthe level of cTnI in the sample has increased from the first sample tothe second sample and predicting a favorable outcome in the humansubject if the level of cTnI in the sample has remained unchanged or hasdecreased from the first sample to the second sample.

In some embodiments of the above described method, the first sample istaken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of after an injury to the head. Inanother embodiment, the first sample is taken from the subject withinabout 30 minutes after an injury to the head. In another embodiment, thefirst sample is taken from the subject within about 1 hour after aninjury to the head. In some embodiments of the above method, the firstsample is taken from the subject within about 2 hours after an injury tothe head. In some embodiments of the above method, the first sample istaken from the subject within about 3 hours after an injury to the head.In some embodiments of the above method, the first sample is taken fromthe subject within about 4 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 5 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 6 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 7 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 8 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 9 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 10 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 11 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 12 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 13 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 14 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 15 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 16 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 17 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 18 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 19 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 20 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 21 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 22 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 23 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 24 hours after an injury to the head.

In some embodiments of the above method, the method predicts a favorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 6 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 6 months after an injury to the head.

In some embodiments of the above method, the subject has a normal headCT scan.

In some embodiments of the above method, the subject has an abnormalhead CT scan.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments of the above method, the subject has an ExtendedGlasgow Outcome (GOSE) score of 5 or less. In some embodiments of theabove method, the GOSE score is obtained before or after the assay isperformed.

In some embodiments of the above method, the amount of cTnI in the firstsample is from about 1.0 to about 50 pg/mL.

In some embodiments of the above method, the amount of cTnI in thesecond sample is from about 1.0 to about 50 pg/mL.

In some embodiments of the above method, the level of cTnI decreases orincrease by at least an absolute amount from the first sample to thesecond sample. More specifically, the absolute amount is determined byan assay having a sensitivity of between at least about 80% to 100% anda specificity of between at least about 45% to 100%. In some embodimentsof the above method, the absolute amount is determined by an assayhaving a sensitivity of at least about 82.4% and a specificity of atleast about 69.5%. In some embodiments of the above method, the absoluteamount is between at least about 1 pg/mL to about 50 pg/mL.

In some embodiments of the above method, the absolute amount is about5.6 pg/mL.

In some embodiments of the above method, the subject (having a mild TBIor a moderate, severe, or moderate to severe TBI) that is assessed ashaving an unfavorable outcome is treated with a traumatic brain injurytreatment. In some embodiments of the above method, the traumatic braininjury treatment for a subject suffering from a mild TBI can involvehaving the subject rest for a certain period of time, abstain fromphysical activities for a certain period of time, administration of oneor more therapeutics (e.g., drugs to provide relief for a headache ormigraine, etc.) or combinations thereof. In other embodiments of theabove method, the traumatic brain injury treatment for a subjectsuffering from a moderate, severe, or moderate to severe TBI, thetreatment involves the administration of one or more therapeutics (e.g.,drugs such as diuretics, anti-seizure drugs), performing one or moresurgical procedures (e.g., such as removal of a hematoma, repairing askull fracture, decompressive crainiectomy, etc.), receipt or providingof one or more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof.

In some embodiments of the above method, the subject having beenassessed as having an unfavorable outcome may also be monitored. Saidsubject may be monitored regardless of whether the subject is receivinga traumatic brain injury treatment or other treatment (such as one ormore cardioprotective therapies).

The present disclosure is directed to a method for aiding in thediagnosis and evaluation of mild traumatic brain injury in a humansubject, the method comprising: a) performing an assay on samples fromthe human subject to measure or detect a level of cTnI in a first sampleand a second sample, wherein the first sample is taken from the humansubject at a first time point and the second sample is taken from thehuman subject about 1 hours to about 4 hours after the first sample,wherein the samples are biological samples; b) determining whether theamount of cTnI has increased or decreased from the first sample to thesecond sample; and c) confirming the occurrence of moderate to severetraumatic brain injury if the level of cTnI detected has increased fromthe first sample to the second sample and confirming the absence of mildtraumatic brain injury if the level of cTnI detected has remainedunchanged or has decreased from the first sample to the second sample.

The present disclosure is directed to a method for aiding in predictingor predicting the outcome of human subjects having mild traumatic braininjury, the method comprising: a) performing an assay on samples fromthe human subject to measure or detect a level of cTnI in a first sampleand a second sample, wherein the first sample is taken from the humansubject at a first time point within 24 hours after an injury to thehead and the second sample is taken from the human subject about 0 toabout 4 hours after the first sample, wherein the samples are biologicalsamples; b) determining the age of the subject; and c) predicting anunfavorable outcome in the human subject if the level of cTnI in thefirst sample and/or second sample are higher than a reference level ofcTnI and the age of the subject is above a reference age and predictinga favorable outcome in the human subject if the level of cTnI in thefirst sample and second sample are lower than a reference levels of cTnIand/or the age of the subject is below the reference age.

In some embodiments of the above described method, the first sample istaken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of after an injury to the head. Inanother embodiment, the first sample is taken from the subject withinabout 30 minutes after an injury to the head. In another embodiment, thefirst sample is taken from the subject within about 1 hour after aninjury to the head. In some embodiments of the above method, the firstsample is taken from the subject within about 2 hours after an injury tothe head. In some embodiments of the above method, the first sample istaken from the subject within about 3 hours after an injury to the head.In some embodiments of the above method, the first sample is taken fromthe subject within about 4 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 5 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 6 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 7 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 8 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 9 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 10 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 11 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 12 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 13 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 14 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 15 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 16 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 17 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 18 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 19 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 20 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 21 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 22 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 23 hours after an injury to the head. In someembodiments of the above method, the first sample is taken from thesubject within about 24 hours after an injury to the head.

In some embodiments of the above method, the age of the subject is 18 to30 years. In other embodiments of the above method, the age of thesubject is 31 to 50 years. In yet other embodiments of the above method,the age of the subject is 51 to 70 years. In yet other embodiments ofthe above method, the age of the subject is 70 to 100 years. In yetother embodiments of the above method, the age is 18 years or less. Inyet other embodiments of the above method, the age is 19-50 years. Instill further embodiments, the age is 51-70 years. In yet otherembodiments of the above method, the age is greater than 70 years. Inyet other embodiments of the above method, the age of the subject is 20to 30 years. In yet other embodiments of the above method, the age ofthe subject is 31 to 40 years. In yet other embodiments of the abovemethod, the age of the subject is 41 to 50 years. In yet otherembodiments of the above method, the age of the subject is 51 to 60years. In yet other embodiments of the above method, the age of thesubject is 61 to 70 years. In yet other embodiments of the above method,the age of the subject is 71 to 80 years. In yet other embodiments ofthe above method, the age of the subject is 81 to 90 years. In yet otherembodiments of the above method, the age of the subject is 91 to 100years.

In some embodiments of the above method, the method predicts a favorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 1 month after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 3 months after an injury to the head. Insome embodiments of the above method, the method predicts a favorableoutcome of the subject at about 6 months after an injury to the head. Insome embodiments of the above method, the method predicts an unfavorableoutcome of the subject at about 6 months after an injury to the head.

In some embodiments of the above method, the subject has a normal headCT scan.

In some embodiments of the above method, the subject has an abnormalhead CT scan.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether or not the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments of the above method, the subject has an ExtendedGlasgow Outcome (GOSE) score of 5 or less. In some embodiments of theabove method, the GOSE score is obtained before or after the assay isperformed.

In some embodiments of the above method, the subject (having a mild TBIor a moderate, severe, or moderate to severe TBI) that is assessed ashaving an unfavorable outcome is treated with a traumatic brain injurytreatment. In some embodiments of the above method, the traumatic braininjury treatment for a subject suffering from a mild TBI can involvehaving the subject rest for a certain period of time, abstain fromphysical activities for a certain period of time, administration of oneor more therapeutics (e.g., drugs to provide relief for a headache ormigraine, etc.) or combinations thereof. In other embodiments of theabove method, the traumatic brain injury treatment for a subjectsuffering from a moderate, severe, or moderate to severe TBI, thetreatment involves the administration of one or more therapeutics (e.g.,drugs such as diuretics, anti-seizure drugs), performing one or moresurgical procedures (e.g., such as removal of a hematoma, repairing askull fracture, decompressive crainiectomy, etc.), receipt or providingof one or more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof.

In some embodiments of the above method, the subject having beenassessed as having an unfavorable outcome may also be monitored. Saidsubject may be monitored regardless of whether the subject is receivinga traumatic brain injury treatment or other treatment (such as one ormore cardioprotective therapies).

In some embodiments, in any of the above methods, the methods furthercomprise providing a cardioprotective therapy to the subject. Suchcardioprotective treatment can be any cardioprotective treatment knownin the art, optionally including but not limited to one or more of abeta-blocker, a diuretic, an Angiotensin-Converting Enzyme (ACE)inhibitor, a calcium channel blocker, a lipid lowering therapy, astatin, a nitrate, an antiplatelet, an anticlotting agent, ananticoagulation agent or combinations thereof. Such one or morecardioprotective therapies can be given alone or in combination with oneor more traumatic brain injury treatments.

In some embodiments in any of the above methods, the level of cTnI ismeasured by an immunoassay or clinical chemistry assay. In otherembodiments, the level of cTnI in the above methods is measured by:

A. contacting the sample, either simultaneously or sequentially, in anyorder with:

(1) a cTnI-capture antibody, which binds to an epitope on cTnI or cTnIfragment to form a cTnI-capture antibody-cTnI antigen complex, and

(2) a cTnI-detection antibody which includes a detectable label andbinds to an epitope on cTnI that is not bound by the cTnI-captureantibody, to form a cTnI antigen-cTnI-detection antibody complex,

such that a cTnI-capture antibody-cTnI antigen-cTnI-detection antibodycomplex is formed, and

B. measuring the amount or concentration of cTnI in the sample based onthe signal generated by the detectable label in the cTnI-captureantibody-cTnI antigen-cTnI-detection antibody complex.

In some embodiments of any of the above methods, the sample is selectedfrom the group consisting of a whole blood sample, a serum sample, acerebrospinal fluid sample, and a plasma sample. In some embodiments inthe above methods, the sample is obtained after the subject sustained aninjury to the head caused by physical shaking, blunt impact by anexternal mechanical or other force that results in a closed or open headtrauma, one or more falls, explosions or blasts or other types of bluntforce trauma. In yet other embodiments in the above methods, the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin. In someembodiments in the above methods, the chemical or toxin is fire, mold,asbestos, a pesticide, an insecticide, an organic solvent, a paint, aglue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof. In some embodiments in the above methods, thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

In some embodiments of any of the above methods, the methods can becarried out on any subject without regard to factors selected from thegroup consisting of the subject's clinical condition, the subject'slaboratory values, the subject's classification as suffering from mild,moderate, severe, or a moderate to severe traumatic brain injury, thesubject's exhibition of low or high levels of cTnI, and the timing ofany event wherein said subject may have sustained an injury to the head.

In some embodiments, in any of the above methods, the sample is a wholeblood sample.

In some embodiments, in any of the above methods, the sample is a plasmasample.

In some embodiments, in any of the above methods, the sample is a serumsample.

In some embodiments, in any of the above methods, the assay is animmunoassay.

In some embodiments, in any of the above methods, the assay is aclinical chemistry assay.

In some embodiments, in any of the above methods, the assay is a singlemolecule detection assay.

In some embodiments, in any of the above methods, the sample is a wholeblood sample and the assay is an immunoassay. In other embodiments, thesample is a plasma sample and the assay is an immunoassay. In yet otherembodiments, the sample is a serum sample and the assay is animmunoassay. In other embodiments, in the above methods, the sample is awhole blood sample and the assay is a clinical chemistry assay. In otherembodiments, the sample is a plasma sample and the assay is a clinicalchemistry assay. In yet other embodiments, the sample is a serum sampleand the assay is a clinical chemistry. In other embodiments, in theabove methods, the sample is a whole blood sample and the assay is asingle molecule detection assay. In other embodiments, the sample is aplasma sample and the assay is a single molecule detection assay. In yetother embodiments, the sample is a serum sample and the assay is asingle molecule detection assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a box plot of hsTnI assay results by time point.

FIG. 2 shows box plot of hsTnI assay results at Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1) correlated with positive vs.negative CT scan results.

FIG. 3 shows box plots of absolute amount (“absolute delta”) of hsTnIresults (i.e., the absolute difference between Time Point 2 (taken 3 to6 hours after the sample of Time Point 1) and Time Point 1 (taken within0 to 6 hours after head injury)) correlated with positive vs. negativeCT scan results.

FIG. 4 shows box plot of hsTnI assay results at Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 5 shows box plot of absolute amount (“absolute delta”) hsTnI assayresults (i.e., the absolute difference between Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1)) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 6 shows biomarker hsTnI result vs. time from injury based on CTscan results.

FIG. 7 shows biomarker hsTnI result vs. time from injury based onGlasgow Coma Scale (GCS) scores.

FIG. 8 shows a receiver operating characteristic (ROC) analysis of hsTnIlevels correlated with CT status (positive vs. negative CT scan result)in samples taken within about 2 hours of suspected injury. The sample atTime Point 1 is taken within 2 hours of head injury while the sample atTime Point 2 is taken about 3 to about 6 hours after the Time Point 1sample is taken.

FIG. 9 shows a receiver operating characteristic (ROC) analysis of hsTnIlevels correlated with mild vs. moderate/severe TBI GCS scores insamples taken within about 2 hours of suspected injury. The sample atTime Point 1 is taken within 2 hours of head injury while the sample atTime Point 2 is taken about 3 to about 6 hours after the Time Point 1sample is taken.

FIG. 10 shows ROC curve of hsTnI assay results for all of the subjectsat Time Point 1 correlated with positive vs. negative CT scan results.

FIG. 11 shows ROC curve of hsTnI assay results for all of the subjectsat Time Point 1 correlated with mild vs. moderate/severe TBI GCS scores.

FIG. 12 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with CT status(positive vs. negative CT scan result). The sample at Time Point 1 istaken within 2 hours of head injury while the sample at Time Point 2 istaken about 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 13 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with mild vs.moderate/severe TBI GCS scores. The sample at Time Point 1 is takenwithin 2 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 14 shows ROC curve of absolute amount (“absolute delta”) hsTnIassay results for all of the subjects correlated with positive vs.negative CT scan results.

FIG. 15 shows ROC curve of absolute amount (“absolute delta”) hsTnIassay results for all of the subjects correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 16 shows the distribution of hsTnI levels in TBI patients andTrauma control patients (p=0.0001).

FIG. 17 shows the distribution of hsTnI levels in TBI patients havingabnormal head CT and normal head CT scan (p=0.005).

FIG. 18 shows the distribution of hsTnI levels in TBI patients with GCSscores of <13, 13, 14, or 15 (p=0.46).

FIG. 19 shows the distribution of hsTnI levels in TBI patients havingGOSE scores of 1, 3, 4, 5, 6, 7, or 8 at 1 month post-injury (p=0.0001).

FIG. 20 shows the distribution of hsTnI levels in TBI patients havingGOSE scores of 1, 2, 3, 4, 5, 6, 7, or 8 at 3 months post-injury(p=0.02).

FIG. 21 shows the distribution of hsTnI levels in TBI patients havingGOSE scores of 1, 3, 4, 5, 6, 7, or 8 at 6 months post-injury (p=0.11).

FIG. 22 shows the distribution of hsTnI levels in samples taken at 0hours, 4 hours, 24 hours, and 1 month after injury from TBI patientshaving abnormal head CT scan and normal head CT scan.

FIG. 23 shows the distribution of the 4 hour absolute change in hsTnIlevels in TBI patients having GOSE scores at 1 month post-injury of 1,3, 4, 5, 6, 7, or 8.

FIG. 24 shows the distribution of hsTnI levels in TBI patients based onage: 18 years or less, 19 to 50 years, 51 to 70 years, and greater than70 years.

FIG. 25 shows a distribution of hsTnI values in TBI and trauma controlparticipants. TBI participants had higher hsTnI values than traumacontrol participants.

FIG. 26 shows a graphical display of the association between cardiactroponin I values and TBI outcome.

FIG. 27 shows Area Under the Receiver Operator Characteristics (AUROC)analysis for discriminating between subjects with 1 month GOSE<5 versusGOSE>5 using the initial hsTnI levels (AUROC curve=0.7135).

FIG. 28 shows AUROC analysis for discriminating between subjects with 1month GOSE<5 versus GOSE>5 using the 4 hour hsTnI levels (AUROCcurve=0.7715).

FIG. 29 shows AUROC analysis for discriminating between subjects with 1month GOSE<5 versus GOSE>5 using the relative change between the initialand 4 hour hsTnI levels (AUROC curve=0.7070).

FIG. 30 shows AUROC analysis for discriminating between subjects with 1month GOSE<5 versus GOSE>5 using the 24 hour hsTnI levels (AUROCcurve=0.7868).

FIG. 31 shows AUROC analysis for predictive model that includes initialhsTnI levels, 4 hour hsTnI levels, and age (AUROC=0.8408).

DETAILED DESCRIPTION

The present disclosure relates to several discoveries involving the useof cardiac troponin I (cTnI) as a biomarker in connection with traumaticbrain injury (TBI). Cardiac troponin I is well known in the art as ahighly specific biomarker for myocardial injury. In fact, severalcommercial assays are available for measuring cTnI in blood or plasmafor this purpose. However, it is also known in the art that cardiacdysfunction is frequently observed in subjects that have suffered asevere TBI. However, the significance of such cardiac dysfunction inthese subjects traditionally has been poorly understood.

In contrast, the present disclosure provides new and improved methods ofusing levels and/or changes in the levels of cTnI (e.g., by performingan assay to determine the level of cTnI in one or more biologicalsamples and then comparing those level(s) to one or more referencelevel(s)) as an aid in the evaluation, diagnoses and/or stratificationof whether a subject that has suffered an injury or is believed to havesuffered an injury to the head has suffered mild TBI, a moderate TBI, asevere TBI, a moderate to severe TBI or no TBI whatsoever. The methodsdescribed herein can be performed quickly—in as little as 2 hours and upto about 24 hours after an injury or suspected injury to the head. Theuse of cTnI to differentiate between mild, moderate, severe, moderate tosevere or no TBI in this manner is previously unknown. Not only do suchmethods allow a physician to quickly determine and classify (orreclassify) or triage a patient as having a TBI or no TBI, for thosepatients identified or determined to have suffered a TBI, the methodsdescribed herein allow the physician to determine the type of TBI (mildversus moderate, severe, or moderate to severe). The ability to quicklydetermine whether classify a TBI as mild, moderate, severe or moderateto severe allows the physician to development an appropriate course oftreatment (e.g., treatment plan) for the subject. Such a treatment plancan include whether to (1) order one or more additional tests to obtainfurther clinical information about the TBI (e.g., such as a MRI, etc.);(2) begin (continue) monitoring the subject; (3) begin treating thesubject with a traumatic brain injury treatment (and if treatment isbegun, what type of treatment to begin (e.g, one or more therapeutictreatments, protecting the airway, one or more surgical treatments,ordering rest, etc.); (4) begin any cardioprotective treatment toprotect the heart of the subject (such as, optionally, by theadministration of one or more beta-blockers, diuretics,angiotensin-converting inhibitors, calcium channel blockers, lipidlowering therapies, statins, nitrates, antiplatelet therapy,anticlotting agents, anticoagulation agents or combinations thereof, orother cardioprotective agents known in the art); or (5) perform anycombinations of (1)-(4).

Additionally, the present disclosure provides methods of using levels orchanges in the levels of cTnI as an aid in determining whether a headcomputerized tomography (CT) should be performed on a subject that hassuffered or is believed to have suffered a TBI. The methods describedherein can be performed quickly—in as little as 2 hours and up to about24 hours after an injury or suspected injury to the head. The use ofcTnI as an aid in assisting a physician to determine whether or toperform a head CT in subjects that have suffered or believed to havesuffered a TBI is previously not known.

Moreover, the present disclosure also provides methods of using levelsor changes in the levels of cTnI to determine the outcome of subjectssuffering from a mild TBI. Specifically, the methods described hereincan be used to determine whether a subject diagnosed with a mild TBI ismore likely than not to have (1) a favorable outcome (optionally, thefavorable outcome can be that the subject fully recovers and does notcontinue to experience one or more symptoms of a mild TBI); or (2) anunfavorable outcome (optionally, the unfavorable outcome can be that thesubject does not fully recover and does continue to experience one ormore symptoms of a mild TBI).

Alternatively and optionally, a favorable outcome can mean that thesubject is more likely than not to suffer no more than onepost-concussion syndrome symptom as a result of the mild TBI such as:(a) physical difficulties (e.g., headaches, dizziness, fatigue,sensitivity to light noise and light, etc.); (b) cognitive difficulties(e.g., trouble concentration, memory problems, restlessness, etc.); (c)emotional difficulties (e.g., personality changes, irritability,depression, apathy, etc.); or (d) sleep difficulties (e.g., insomnia,etc.). Alternatively and optionally, subject who have an unfavorableoutcome are more likely to suffer from more than one post-concussionsyndrome symptom such as: (a) physical difficulties (e.g., headaches,dizziness, fatigue, sensitivity to light noise and light, etc.); (b)cognitive difficulties (e.g., trouble concentration, memory problems,restlessness, etc.); (c) emotional difficulties (e.g, personalitychanges, irritability, depression, apathy, etc.); (d) sleep difficulties(e.g., insomnia, etc.); or (e) any combinations of (a)-(d)).Alternatively and optionally, an unfavorable outcome can also mean thata subject exhibits one or more symptoms of mild TBI. Alternatively andoptionally, an unfavorable outcome can also mean that the subject'sconditions worsens from mild TBI to moderate, moderate to severe orsevere. Additionally, subjects having a favorable outcome are likely tohave a GOSE score of 5 or greater whereas subjects having an unfavorableoutcome are likely to have a GOSE score of less than 5.

Subjects unlikely to make a full recovery from the mild TBI will likelyneed one or more of additional therapeutic treatment, physical therapyand/or occupational therapy for at least 1 day, 2 days, 3 days, 4 days,5 days 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months 12 months, 13 months, 14 months,15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13years, 14 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40years, 45 years or 50 years after diagnosis of the mild TBI. The use ofcTnI in predicting outcome in mild TBI subjects is previously not known.

Additionally, the present disclosure provides methods of treating atraumatic brain injury. Specifically, the methods involve using levelsand/or changes in the levels of cTnI as described herein (e.g., byperforming an assay to determine the level of cTnI in one or morebiological samples and then comparing those level(s) to one or morereference level(s)) to evaluate, diagnose and/or stratify whether asubject that has suffered an injury to the head or is believed to havesuffered an injury to the head has suffered mild TBI, a moderate TBI,severe TBI, moderate to severe TBI or no TBI. Once a subject has beenidentified, determined, classified or stratified as having a mild TBI ora moderate, severe or moderate to severe TBI, then depending on the typeof TBI (mild versus moderate, severe or moderate to severe), the subjectcan be treated with an appropriate traumatic brain injury treatment. Forexample, for a mild TBI, the traumatic brain injury treatment mayinvolve one or more of having the subject rest for a certain period oftime, abstain from physical activities for a certain period of time,administration of one or more therapeutics (e.g., drugs to providerelief for a headache or migraine, etc.) or combinations thereof. For amoderate, severe, or moderate to severe TBI, the traumatic brain injurytreatment may involve administration of one or more therapeutics (e.g.,drugs such as diuretics, anti-seizure drugs), performing one or moresurgical procedures (e.g., such as removal of a hematoma, repairing askull fracture, decompressive crainiectomy, etc.), receipt of one ormore therapies (such as rehabilitation, physical therapy, occupationaltherapy, cognitive behavioral therapy, anger management, etc.) orcombinations thereof. Optionally, such methods may also involveproviding one or more cardioprotective therapies. Such cardioprotectivetherapies can be administered in combination with the treatments for theTBI or alone without any TBI treatment, depending on the circumstances.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI).Furthermore, in order to confirm that the changes in amounts or levelscTnI in the methods described herein are attributable to a head injuryor a suspected injury to the head of a subject and not the result of anacute cardiac syndrome (such as a myocardial infarction, heart failure,etc.), a physician or other healthcare provider could conduct or performone or more additional tests or procedures to confirm the absence of anacute cardiac syndrome. Such additional tests or procedures include oneor more of an electrocardiogram, a complete blood cell (CBC) count, acomprehensive metabolic panel, a lipid profile (e.g., to determine HDL,LDL, triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

Section headings as used in this section and the entire disclosureherein are merely for organizational purposes and are not intended to belimiting.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentdisclosure. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity matured antibody” is used herein to refer to an antibody withone or more alterations in one or more CDRs, which result in animprovement in the affinity (i.e., K_(D), k_(d) or k_(a)) of theantibody for a target antigen compared to a parent antibody, which doesnot possess the alteration(s). Exemplary affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.A variety of procedures for producing affinity matured antibodies isknown in the art, including the screening of a combinatory antibodylibrary that has been prepared using bio-display. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); and Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity-enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

“Antibody” and “antibodies” as used herein refers to monoclonalantibodies, multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized), animal antibodies such as,but not limited to, a bird (for example, a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate(for example, a monkey, a chimpanzee, etc.), recombinant antibodies,chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments, F(ab′)2fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11): 1290-1297 (2007) and PCT InternationalApplication WO 2001/058956, the contents of each of which are hereinincorporated by reference), and functionally active epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, namely, molecules that contain ananalyte-binding site. Immunoglobulin molecules can be of any type (forexample, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity sake, anantibody against an analyte is frequently referred to herein as beingeither an “anti-analyte antibody” or merely an “analyte antibody” (e.g.,an anti-cardiac troponin I antibody or a cardiac troponin I antibody).

“Antibody fragment” as used herein refers to a portion of an intactantibody comprising the antigen-binding site or variable region. Theportion does not include the constant heavy chain domains (i.e., CH2,CH3, or CH4, depending on the antibody isotype) of the Fc region of theintact antibody. Examples of antibody fragments include, but are notlimited to, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv)molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs ofthe light-chain variable domain, single-chain polypeptides containingonly one heavy chain variable region, and single-chain polypeptidescontaining the three CDRs of the heavy chain variable region.

The “area under curve” or “AUC” refers to area under a ROC curve. AUCunder a ROC curve is a measure of accuracy. An AUC of 1 represents aperfect test, whereas an AUC of 0.5 represents an insignificant test. Apreferred AUC may be at least approximately 0.700, at leastapproximately 0.750, at least approximately 0.800, at leastapproximately 0.850, at least approximately 0.900, at leastapproximately 0.910, at least approximately 0.920, at leastapproximately 0.930, at least approximately 0.940, at leastapproximately 0.950, at least approximately 0.960, at leastapproximately 0.970, at least approximately 0.980, at leastapproximately 0.990, or at least approximately 0.995.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support. One example of a bead or particleis a microparticle. Microparticles that can be used herein can be anytype known in the art. For example, the bead or particle can be amagnetic bead or magnetic particle. Magnetic beads/particles may beferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The microparticles can be of any size thatwould work in the methods described herein, e.g., from about 0.75 toabout 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3nm.

“Binding protein” is used herein to refer to a monomeric or multimericprotein that binds to and forms a complex with a binding partner, suchas, for example, a polypeptide, an antigen, a chemical compound or othermolecule, or a substrate of any kind. A binding protein specificallybinds a binding partner. Binding proteins include antibodies, as well asantigen-binding fragments thereof and other various forms andderivatives thereof as are known in the art and described herein below,and other molecules comprising one or more antigen-binding domains thatbind to an antigen molecule or a particular site (epitope) on theantigen molecule. Accordingly, a binding protein includes, but is notlimited to, an antibody a tetrameric immunoglobulin, an IgG molecule, anIgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

“Bispecific antibody” is used herein to refer to a full-length antibodythat is generated by quadroma technology (see Milstein et al., Nature,305(5934): 537-540 (1983)), by chemical conjugation of two differentmonoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628-631(1985)), or by knob-into-hole or similar approaches, which introducemutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci.USA, 90(14): 6444-6448 (1993)), resulting in multiple differentimmunoglobulin species of which only one is the functional bispecificantibody. A bispecific antibody binds one antigen (or epitope) on one ofits two binding arms (one pair of HC/LC), and binds a different antigen(or epitope) on its second arm (a different pair of HC/LC). By thisdefinition, a bispecific antibody has two distinct antigen-binding arms(in both specificity and CDR sequences), and is monovalent for eachantigen to which it binds to.

As used herein, the terms “cardiac troponin I”, “cTnI” or “troponin I”as used interchangeably herein, refers to one of two unique forms ofcardiac troponin (the other unique form being cardiac troponin T (alsoreferred to as “cTnT”)), released into the blood from cardiac muscle forwhich several species may exist in the blood. Not only does the term“cardiac troponin I” or “cTnI” include the full-length version of thisform but it also includes: (1) various complexes of cTnI (namely, witheach other and/or with cardiac troponin C (cTnC)); (2) fragments of cTnIwhich result from proteolytic degradation; (3) phosphorylated andoxidized forms of cTnI (See, for example, U.S. Pat. No. 6,991,907, thecontents of which are herein incorporated by reference); and (4) anyisoforms of cTnI.

In some embodiments, the methods of the present disclosure allow for thedetection and/or determination of concentration of one or more of thevarious forms of cTnI in a sample as a separate entity, e.g., complexedcTnI, free cTnI (e.g., such as fully length, fragments, isoforms, etc.),muddied cTnI (e.g., oxidized or phosphorylated), and, optionally,provides a concentration for the cTnI in the biological sample.

More specifically, in some embodiments, the disclosure described hereinemploys highly sensitivity assays that allow for the detection andquantification of cTnI at levels 10- to 100-fold lower than levelsmeasured by traditional troponin assays (e.g., immunoassays) known inthe art. More specifically, assays are defined as high sensitivity(e.g., high sensitivity assays for troponin) if such assays meet atleast the following two conditions: 1) a coefficient of variance lessthan 10% at the 99th percentile value of the reference healthypopulation and 2) concentrations above the assay's limit of detectionare measurable in greater than 50% of healthy individuals (See, Apple FS, et al., Clin Chem., 58:54-61 (2012), the contents of which are herebyincorporated by reference). Examples of assays known in the art thatallow for the high-sensitive detection of troponin include thoseavailable from Quanterix (Simoa Human Troponin-I immunoassay) forresearch use only as well as those described in U.S. Pat. No. 9,182,405,the contents of which are hereby incorporated by reference.

“Cardiac Troponin I status” or “cTnI status” as used interchangeablyherein can mean either the level or amount of cardiac troponin I at apoint in time (such as with a single measure of troponin I), the levelor amount of cardiac troponin I associated with monitoring (such as witha repeat test on a subject to identify an increase or decrease incardiac troponin I amount), the level or amount of cardiac troponin Iassociated with treatment for traumatic brain injury (whether a primarybrain injury and/or a secondary brain injury) or combinations thereof.

“CDR” is used herein to refer to the “complementarity determiningregion” within an antibody variable sequence. There are three CDRs ineach of the variable regions of the heavy chain and the light chain.Proceeding from the N-terminus of a heavy or light chain, these regionsare denoted “CDR1”, “CDR2”, and “CDR3”, for each of the variableregions. The term “CDR set” as used herein refers to a group of threeCDRs that occur in a single variable region that binds the antigen. Anantigen-binding site, therefore, may include six CDRs, comprising theCDR set from each of a heavy and a light chain variable region. Apolypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) maybe referred to as a “molecular recognition unit.” Crystallographicanalyses of antigen-antibody complexes have demonstrated that the aminoacid residues of CDRs form extensive contact with bound antigen, whereinthe most extensive antigen contact is with the heavy chain CDR3. Thus,the molecular recognition units may be primarily responsible for thespecificity of an antigen-binding site. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as “Kabat CDRs”.Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917(1987); and Chothia et al., Nature, 342: 877-883 (1989)) found thatcertain sub-portions within Kabat CDRs adopt nearly identical peptidebackbone conformations, despite having great diversity at the level ofamino acid sequence. These sub-portions were designated as “L1”, “L2”,and “L3”, or “H1”, “H2”, and “H3”, where the “L” and the “H” designatethe light chain and the heavy chain regions, respectively. These regionsmay be referred to as “Chothia CDRs”, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995),and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDRboundary definitions may not strictly follow one of the herein systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat- or Chothia-defined CDRs.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection or conjugate a calibrator, a control,a sensitivity panel, a container, a buffer, a diluent, a salt, anenzyme, a co-factor for an enzyme, a detection reagent, a pretreatmentreagent/solution, a substrate (e.g., as a solution), a stop solution,and the like that can be included in a kit for assay of a test sample,such as a patient urine, whole blood, serum or plasma sample, inaccordance with the methods described herein and other methods known inthe art. Some components can be in solution or lyophilized forreconstitution for use in an assay.

“Correlated to” as used herein refers to compared to.

“CT scan” as used herein refers to a computerized tomography (CT) scan.A CT scan combines a series of X-ray images taken from different anglesand uses computer processing to create cross-sectional images, orslices, of the bones, blood vessels and soft tissues inside your body.The CT scan may use X-ray CT, positron emission tomography (PET),single-photon emission computed tomography (SPECT), computed axialtomography (CAT scan), or computer aided tomography. The CT scan may bea conventional CT scan or a spiral/helical CT scan. In a conventional CTscan, the scan is taken slice by slice and after each slice the scanstops and moves down to the next slice, e.g., from the top of theabdomen down to the pelvis. The conventional CT scan requires patientsto hold their breath to avoid movement artefact. The spiral/helical CTscan is a continuous scan which is taken in a spiral fashion and is amuch quicker process where the scanned images are contiguous.

“Derivative” of an antibody as used herein may refer to an antibodyhaving one or more modifications to its amino acid sequence whencompared to a genuine or parent antibody and exhibit a modified domainstructure. The derivative may still be able to adopt the typical domainconfiguration found in native antibodies, as well as an amino acidsequence, which is able to bind to targets (antigens) with specificity.Typical examples of antibody derivatives are antibodies coupled to otherpolypeptides, rearranged antibody domains, or fragments of antibodies.The derivative may also comprise at least one further compound, e.g., aprotein domain, said protein domain being linked by covalent ornon-covalent bonds. The linkage can be based on genetic fusion accordingto the methods known in the art. The additional domain present in thefusion protein comprising the antibody may preferably be linked by aflexible linker, advantageously a peptide linker, wherein said peptidelinker comprises plural, hydrophilic, peptide-bonded amino acids of alength sufficient to span the distance between the C-terminal end of thefurther protein domain and the N-terminal end of the antibody or viceversa. The antibody may be linked to an effector molecule having aconformation suitable for biological activity or selective binding to asolid support, a biologically active substance (e.g., a cytokine orgrowth hormone), a chemical agent, a peptide, a protein, or a drug, forexample.

“Determined by an assay” is used herein to refer to the determination ofa reference level by any appropriate assay. The determination of areference level may, in some embodiments, be achieved by an assay of thesame type as the assay that is to be applied to the sample from thesubject (for example, by an immunoassay, clinical chemistry assay, asingle molecule detection assay, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS)). The determination of a reference level may, in someembodiments, be achieved by an assay of the same type and under the sameassay conditions as the assay that is to be applied to the sample fromthe subject. As noted herein, this disclosure provides exemplaryreference levels (e.g., calculated by comparing reference levels atdifferent time points). It is well within the ordinary skill of one inthe art to adapt the disclosure herein for other assays to obtainassay-specific reference levels for those other assays based on thedescription provided by this disclosure. For example, a set of trainingsamples comprising samples obtained from human subjects known to havesustained an injury to the head (and more particularly, samples obtainedfrom human subjects known to have sustained a (i) mild TBI; and/or (ii)moderate, severe, or moderate to severe TBI and samples obtained fromhuman subjects known not to have sustained an injury to the head may beused to obtain assay-specific reference levels. It will be understoodthat a reference level “determined by an assay” and having a recitedlevel of “sensitivity” and/or “specificity” is used herein to refer to areference level which has been determined to provide a method of therecited sensitivity and/or specificity when said reference level isadopted in the methods of the disclosure. It is well within the ordinaryskill of one in the art to determine the sensitivity and specificityassociated with a given reference level in the methods of thedisclosure, for example by repeated statistical analysis of assay datausing a plurality of different possible reference levels.

Practically, when discriminating between a subject as having a traumaticbrain injury or not having a traumatic brain injury or a subject ashaving a a mild versus a moderate, severe, or moderate to severetraumatic brain injury, the skilled person will balance the effect ofraising a cutoff on sensitivity and specificity. Raising or lowering acutoff will have a well-defined and predictable impact on sensitivityand specificity, and other standard statistical measures. It is wellknown that raising a cutoff will improve specificity but is likely toworsen sensitivity (proportion of those with disease who test positive).In contrast, lowering a cutoff will improve sensitivity but will worsenspecificity (proportion of those without disease who test negative). Theramifications for detecting traumatic brain injury or determining a mildversus moderate, severe, or moderate to severe traumatic brain injurywill be readily apparent to those skilled in the art. In discriminatingwhether a subject has or does not have a traumatic brain injury or amild versus a moderate, severe, or moderate to severe traumatic braininjury, the higher the cutoff, specificity improves as more truenegatives (i.e., subjects not having a traumatic brain injury, nothaving a mild traumatic brain injury, not have a moderate traumaticbrain injury, not having a severe traumatic brain injury or not having amoderate to severe traumatic brain injury) are distinguished from thosehaving a traumatic brain injury, a mild traumatic brain injury, amoderate traumatic brain injury, a severe traumatic brain injury or amoderate to severe traumatic brain injury. But at the same time, raisingthe cutoff decreases the number of cases identified as positive overall,as well as the number of true positives, so the sensitivity mustdecrease. Conversely, the lower the cutoff, sensitivity improves as moretrue positives (i.e., subjects having a traumatic brain injury, having amild traumatic brain injury, having a moderate traumatic brain injury,having a severe traumatic brain injury or having a moderate to severetraumatic brain injury) are distinguished from those who do not have atraumatic brain injury, a mild traumatic brain injure, a moderatetraumatic brain injury, a severe traumatic brain injury or a moderate tosevere traumatic brain injury. But at the same time, lowering the cutoffincreases the number of cases identified as positive overall, as well asthe number of false positives, so the specificity must decrease.

Generally, a high sensitivity value helps one of skill rule out diseaseor condition (such as a traumatic brain injury, mild traumatic braininjury, moderate traumatic brain injury, severe traumatic brain injuryor moderate to severe traumatic brain injury), and a high specificityvalue helps one of skill rule in disease or condition. Whether one ofskill desires to rule out or rule in disease depends on what theconsequences are for the patient for each type of error. Accordingly,one cannot know or predict the precise balancing employed to derive atest cutoff without full disclosure of the underlying information on howthe value was selected. The balancing of sensitivity against specificityand other factors will differ on a case-by-case basis. This is why it issometimes preferable to provide alternate cutoff (e.g., reference)values so a physician or practitioner can choose.

“Drugs of abuse” is used herein to refer to one or more additivesubstances (such as a drug) taken for non-medical reasons (such as for,example, recreational and/or mind-altering effects). Excessiveoverindulgence, use or dependence of such drugs of abuse is oftenreferred to as “substance abuse”. Examples of drugs of abuse includealcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens(such as ketamine, mescaline (peyote), PCP, psilocybin, DMT and/or LSD),methaqualone, opioids, amphetamines (including methamphetamines),anabolic steroids, inhalants (namely, substances which contain volatilesubstances that contain psychoactive properties such as, for example,nitrites, spray paints, cleaning fluids, markers, glues, etc.) andcombinations thereof.

“Dual-specific antibody” is used herein to refer to a full-lengthantibody that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly, a dual-specific binding protein has twoidentical antigen binding arms, with identical specificity and identicalCDR sequences, and is bivalent for each antigen to which it binds.

“Dual variable domain” is used herein to refer to two or more antigenbinding sites on a binding protein, which may be divalent (two antigenbinding sites), tetravalent (four antigen binding sites), or multivalentbinding proteins. DVDs may be monospecific, i.e., capable of binding oneantigen (or one specific epitope), or multispecific, i.e., capable ofbinding two or more antigens (e.g., two or more epitopes of the sametarget antigen molecule or two or more epitopes of different targetantigens). A preferred DVD binding protein comprises two heavy chain DVDpolypeptides and two light chain DVD polypeptides and is referred to asa “DVD immunoglobulin” or “DVD-Ig.” Such a DVD-Ig binding protein isthus tetrameric and reminiscent of an IgG molecule, but provides moreantigen binding sites than an IgG molecule. Thus, each half of atetrameric DVD-Ig molecule is reminiscent of one half of an IgG moleculeand comprises a heavy chain DVD polypeptide and a light chain DVDpolypeptide, but unlike a pair of heavy and light chains of an IgGmolecule that provides a single antigen binding domain, a pair of heavyand light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of a DVD-Ig binding protein may be derivedfrom a donor (“parental”) monoclonal antibody and thus comprises a heavychain variable domain (VH) and a light chain variable domain (VL) with atotal of six CDRs involved in antigen binding per antigen binding site.Accordingly, a DVD-Ig binding protein that binds two different epitopese.g., two different epitopes of two different antigen molecules or twodifferent epitopes of the same antigen molecule) comprises an antigenbinding site derived from a first parental monoclonal antibody and anantigen binding site of a second parental monoclonal antibody.

A description of the design, expression, and characterization of DVD-Igbinding molecules is provided in PCT Publication No. WO 2007/024715,U.S. Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297(2007). A preferred example of such DVD-Ig molecules comprises a heavychain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a heavy chain constant domain, X1 is alinker with the proviso that it is not CH1, X2 is an Fc region, and n is0 or 1, but preferably 1; and a light chain that comprises thestructural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain, X1 is a linker with the proviso that itis not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, butpreferably 1. Such a DVD-Ig may comprise two such heavy chains and twosuch light chains, wherein each chain comprises variable domains linkedin tandem without an intervening constant region between variableregions, wherein a heavy chain and a light chain associate to formtandem functional antigen binding sites, and a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1, VD2, VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate to formthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein not only binds thesame target molecules bound by its parental monoclonal antibodies, butalso possesses one or more desirable properties of one or more of itsparental monoclonal antibodies. Preferably, such an additional propertyis an antibody parameter of one or more of the parental monoclonalantibodies. Antibody parameters that may be contributed to a DVD-Igbinding protein from one or more of its parental monoclonal antibodiesinclude, but are not limited to, antigen specificity, antigen affinity,potency, biological function, epitope recognition, protein stability,protein solubility, production efficiency, immunogenicity,pharmacokinetics, bioavailability, tissue cross reactivity, andorthologous antigen binding.

A DVD-Ig binding protein binds at least one epitope of cardiac troponinI. Non-limiting examples of a DVD-Ig binding protein include a DVD-Igbinding protein that binds one or more epitopes of cardiac troponin I, aDVD-Ig binding protein that binds an epitope of a human cardiac troponinI and an epitope of cardiac troponin I of another species (for example,mouse), and a DVD-Ig binding protein that binds an epitope of a humancardiac troponin I and an epitope of another target molecule.

“Dynamic range” as used herein refers to range over which an assayreadout is proportional to the amount of target molecule or analyte inthe sample being analyzed.

“Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s)on any molecule that is recognized and can bind to a complementarysite(s) on its specific binding partner. The molecule and specificbinding partner are part of a specific binding pair. For example, anepitope can be on a polypeptide, a protein, a hapten, a carbohydrateantigen (such as, but not limited to, glycolipids, glycoproteins orlipopolysaccharides), or a polysaccharide. Its specific binding partnercan be, but is not limited to, an antibody.

“Fragment antigen-binding fragment” or “Fab fragment” as used hereinrefers to a fragment of an antibody that binds to antigens and thatcontains one antigen-binding site, one complete light chain, and part ofone heavy chain. Fab is a monovalent fragment consisting of the VL, VH,CL and CH1 domains. Fab is composed of one constant and one variabledomain of each of the heavy and the light chain. The variable domaincontains the paratope (the antigen-binding site), comprising a set ofcomplementarity determining regions, at the amino terminal end of themonomer. Each arm of the Y thus binds an epitope on the antigen. Fabfragments can be generated such as has been described in the art, e.g.,using the enzyme papain, which can be used to cleave an immunoglobulinmonomer into two Fab fragments and an Fc fragment, or can be produced byrecombinant means.

“F(ab′)₂ fragment” as used herein refers to antibodies generated bypepsin digestion of whole IgG antibodies to remove most of the Fc regionwhile leaving intact some of the hinge region. F(ab′)₂ fragments havetwo antigen-binding F(ab) portions linked together by disulfide bonds,and therefore are divalent with a molecular weight of about 110 kDa.Divalent antibody fragments (F(ab′)₂ fragments) are smaller than wholeIgG molecules and enable a better penetration into tissue thusfacilitating better antigen recognition in immunohistochemistry. The useof F(ab′)₂ fragments also avoids unspecific binding to Fc receptor onlive cells or to Protein A/G. F(ab′)₂ fragments can both bind andprecipitate antigens.

“Framework” (FR) or “Framework sequence” as used herein may mean theremaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems (for example, see above), the meaning of a framework sequence issubject to correspondingly different interpretations. The six CDRs(CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavychain) also divide the framework regions on the light chain and theheavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on eachchain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2and FR3, and CDR3 between FR3 and FR4. Without specifying the particularsub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred byothers, represents the combined FRs within the variable region of asingle, naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain FR sequences are known in the art thatcan be used as heavy chain and light chain “acceptor” frameworksequences (or simply, “acceptor” sequences) to humanize a non-humanantibody using techniques known in the art. In one embodiment, humanheavy chain and light chain acceptor sequences are selected from theframework sequences listed in publicly available databases such asV-base (hypertext transfer protocol://vbase.mrc-cpe.cam.ac.uk/) or inthe international ImMunoGeneTics® (IMGT®) immunogenetics informationsystem (hypertext transferprotocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).

“Functional antigen binding site” as used herein may mean a site on abinding protein (e.g., an antibody) that is capable of binding a targetantigen. The antigen binding affinity of the antigen binding site maynot be as strong as the parent binding protein, e.g., parent antibody,from which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating protein, e.g., antibody, binding to an antigen. Moreover,the antigen binding affinity of each of the antigen binding sites of amultivalent protein, e.g., multivalent antibody, herein need not bequantitatively the same.

“Glasgow Coma Scale” or “GCS” as used herein refers to a 15 point scalefor estimating and categorizing the outcomes of brain injury on thebasis of overall social capability or dependence on others. The testmeasures the motor response, verbal response and eye opening responsewith these values: I. Motor Response (6—Obeys commands fully;5—Localizes to noxious stimuli; 4—Withdraws from noxious stimuli;3—Abnormal flexion, i.e., decorticate posturing; 2—Extensor response,i.e., decerebrate posturing; and 1—No response); II. Verbal Response(5—Alert and Oriented; 4—Confused, yet coherent, speech; 3—Inappropriatewords and jumbled phrases consisting of words; 2—Incomprehensiblesounds; and 1—No sounds); and III. Eye Opening (4—Spontaneous eyeopening; 3—Eyes open to speech; 2—Eyes open to pain; and 1—No eyeopening). The final score is determined by adding the values ofI+II+III. The final score can be categorized into four possible levelsfor survival, with a lower number indicating a more severe injury and apoorer prognosis: Mild (13-15); Moderate Disability (9-12) (Loss ofconsciousness greater than 30 minutes; Physical or cognitive impairmentswhich may or may resolve: and Benefit from Rehabilitation); SevereDisability (3-8) (Coma: unconscious state. No meaningful response, novoluntary activities); and Vegetative State (Less Than 3) (Sleep wakecycles; Arousal, but no interaction with environment; No localizedresponse to pain). Moderate brain injury is defined as a brain injuryresulting in a loss of consciousness from 20 minutes to 6 hours and aGlasgow Coma Scale of 9 to 12. Severe brain injury is defined as a braininjury resulting in a loss of consciousness of greater than 6 hours anda Glasgow Coma Scale of 3 to 8.

“Glasgow Outcome Scale” as used herein refers to a global scale forfunctional outcome that rates patient status into one of fivecategories: Dead, Vegetative State, Severe Disability, ModerateDisability or Good Recovery.

“Extended Glasgow Outcome Scale” or “GOSE” as used interchangeablyherein provides more detailed categorization into eight categories bysubdividing the categories of severe disability, moderate disability andgood recovery into a lower and upper category as shown in Table 1.

TABLE 1 1 Death D 2 Vegetative VX Condition of unawareness with onlyreflex state responses but with periods of spontaneous eye opening 3Lower severe SD− Patient who is dependent for daily support disabilityfor mental or physical disability, usually a 4 Upper severe SD+combination of both. If the patient can be left disability alone formore than 8 hours at home it is upper level of SD, if not then it is lowlevel of SD. 5 Lower MD− Patients have some disability such as moderateaphasia, hemiparesis or epilepsy and/or disability deficits of memory orpersonality but are 6 Upper MD+ able to look after themselves. They aremoderate independent at home but dependent disability outside. If theyare able to return to work even with special arrangement it is upperlevel of MD, if not then it is low level of MD. 7 Lower good GR−Resumption of normal life with the capacity recovery to work even ifpre-injury status has not been 8 Upper good GR+ achieved. Some patientshave minor recovery neurological or psychological deficits. If thesedeficits are not disabling then it is upper level of GR, if disablingthen it is lower level of GR.

“Humanized antibody” is used herein to describe an antibody thatcomprises heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or VL sequence has been altered to be more “human-like,” i.e.,more similar to human germline variable sequences. A “humanizedantibody” is an antibody or a variant, derivative, analog, or fragmentthereof, which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of a non-human antibody CDR. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. In an embodiment, a humanized antibody also comprises at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsthe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

A humanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE, and any isotype, includingwithout limitation IgG1, IgG2, IgG3, and IgG4. A humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well-known in the art.

The framework regions and CDRs of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion, and/or deletion of at least one amino acid residue so thatthe CDR or framework residue at that site does not correspond to eitherthe donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence.

“Hyperacute” as used herein refers to extremely acute or within a courseof about 2 hours of the injury or suspected injury to the head.Hyperacute is within an early stage, e.g., a hyperacute biomarker is anearly biomarker, such as cTnI, that can be used to assess injury orsuspected injury within the early stage of about 2 hours of injury orsuspected injury.

“Identical” or “identity,” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are the same over aspecified region. The percentage can be calculated by optimally aligningthe two sequences, comparing the two sequences over the specifiedregion, determining the number of positions at which the identicalresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the specified region, and multiplying the result by 100to yield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of the single sequence are included in thedenominator but not the numerator of the calculation.

“Injury to the head” or “head injury” as used interchangeably herein,refers to any trauma to the scalp, skull, or brain. Such injuries mayinclude only a minor bump on the skull or may be a serious brain injury.Such injuries include primary injuries to the brain and/or secondaryinjuries to the brain. Primary brain injuries occur during the initialinsult and result from displacement of the physical structures of thebrain. More specifically, a primary brain injury is the physical damageto parenchyma (tissue, vessels) that occurs during the traumatic event,resulting in shearing and compression of the surrounding brain tissue.Secondary brain injuries occur subsequent to the primary injury and mayinvolve an array of cellular processes. More specifically, a secondarybrain injury refers to the changes that evolve over a period of time(from hours to days) after the primary brain injury. It includes anentire cascade of cellular, chemical, tissue, or blood vessel changes inthe brain that contribute to further destruction of brain tissue.

An injury to the head can be either closed or open (penetrating). Aclosed head injury refers to a trauma to the scalp, skull or brain wherethere is no penetration of the skull by a striking object. An open headinjury refers a trauma to the scalp, skull or brain where there ispenetration of the skull by a striking object. An injury to the head maybe caused by physical shaking of a person, by blunt impact by anexternal mechanical or other force that results in a closed or open headtrauma (e.g., vehicle accident such as with an automobile, plane, train,etc.; blow to the head such as with a baseball bat, or from a firearm),a cerebral vascular accident (e.g., stroke), one or more falls (e.g., asin sports or other activities), explosions or blasts (collectively,“blast injuries”) and by other types of blunt force trauma.Alternatively, an injury to the head may be caused by the ingestionand/or exposure to a chemical, toxin or a combination of a chemical andtoxin. Examples of such chemicals and/or toxins include fires, molds,asbestos, pesticides and insecticides, organic solvents, paints, glues,gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organicmetals (such as methyl mercury, tetraethyl lead and organic tin) and/orone or more drugs of abuse. Alternatively, an injury to the head may becaused as a result of a subject suffering from an autoimmune disease, ametabolic disorder, a brain tumor, one or more viruses, meningitis,hydrocephalus, hypoxia or any combinations thereof. In some cases, it isnot possible to be certain whether any such event or injury has occurredor taken place. For example, there may be no history on a patient orsubject, the subject may be unable to speak, the subject may be aware ofwhat events they were exposed to, etc. Such circumstances are describedherein as the subject “may have sustained an injury to the head.” Incertain embodiments herein, the closed head injury does not include andspecifically excludes a cerebral vascular accident, such as stroke.

“Isolated polynucleotide” as used herein may mean a polynucleotide(e.g., of genomic, cDNA, or synthetic origin, or a combination thereof)that, by virtue of its origin, the isolated polynucleotide is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

“Label” and “detectable label” as used herein refer to a moiety attachedto an antibody or an analyte to render the reaction between the antibodyand the analyte detectable, and the antibody or analyte so labeled isreferred to as “detectably labeled.” A label can produce a signal thatis detectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromagens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like.Representative examples of labels include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety, itself, may not be detectable but may become detectable uponreaction with yet another moiety. Use of the term “detectably labeled”is intended to encompass such labeling.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as 3H,14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and153Sm), an enzymatic label (such as horseradish peroxidase, alkalineperoxidase, glucose 6-phosphate dehydrogenase, and the like), achemiluminescent label (such as acridinium esters, thioesters, orsulfonamides; luminol, isoluminol, phenanthridinium esters, and thelike), a fluorescent label (such as fluorescein (e.g., 5-fluorescein,6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein,6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluoresceinisothiocyanate, and the like)), rhodamine, phycobiliproteins,R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmiumselenide), a thermometric label, or an immuno-polymerase chain reactionlabel. An introduction to labels, labeling procedures and detection oflabels is found in Polak and Van Noorden, Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and inHaugland, Handbook of Fluorescent Probes and Research Chemicals (1996),which is a combined handbook and catalogue published by MolecularProbes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA(see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,and 5,352,803, which are hereby incorporated by reference in theirentireties). An acridinium compound can be used as a detectable label ina homogeneous chemiluminescent assay (see, e.g., Adamczyk et al.,Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg.Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem.Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782(2003)).

In one aspect, the acridinium compound is an acridinium-9-carboxamide.Methods for preparing acridinium 9-carboxamides are described inMattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al.,J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999);Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly etal., In Luminescence Biotechnology: Instruments and Applications; Dyke,K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524and 5,783,699 (each of which is incorporated herein by reference in itsentirety for its teachings regarding same).

Another example of an acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

“Linking sequence” or “linking peptide sequence” refers to a natural orartificial polypeptide sequence that is connected to one or morepolypeptide sequences of interest (e.g., full-length, fragments, etc.).The term “connected” refers to the joining of the linking sequence tothe polypeptide sequence of interest. Such polypeptide sequences arepreferably joined by one or more peptide bonds. Linking sequences canhave a length of from about 4 to about 50 amino acids. Preferably, thelength of the linking sequence is from about 6 to about 30 amino acids.Natural linking sequences can be modified by amino acid substitutions,additions, or deletions to create artificial linking sequences. Linkingsequences can be used for many purposes, including in recombinant Fabs.Exemplary linking sequences include, but are not limited to: (i)Histidine (His) tags, such as a 6× His tag, which has an amino acidsequence of HHHHHH (SEQ ID NO:2), are useful as linking sequences tofacilitate the isolation and purification of polypeptides and antibodiesof interest; (ii) Enterokinase cleavage sites, like His tags, are usedin the isolation and purification of proteins and antibodies ofinterest. Often, enterokinase cleavage sites are used together with Histags in the isolation and purification of proteins and antibodies ofinterest. Various enterokinase cleavage sites are known in the art.Examples of enterokinase cleavage sites include, but are not limited to,the amino acid sequence of DDDDK (SEQ ID NO:3) and derivatives thereof(e.g., ADDDDK (SEQ ID NO:4), etc.); (iii) Miscellaneous sequences can beused to link or connect the light and/or heavy chain variable regions ofsingle chain variable region fragments. Examples of other linkingsequences can be found in Bird et al., Science 242: 423-426 (1988);Huston et al., PNAS USA 85: 5879-5883 (1988); and McCafferty et al.,Nature 348: 552-554 (1990). Linking sequences also can be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. In the context of the present disclosure, the monoclonalantibody, for example, can contain a linking sequence, such as a Histag, an enterokinase cleavage site, or both.

“Magnetic resonance imaging” or “MRI” as used interchangeably hereinrefers to a medical imaging technique used in radiology to form picturesof the anatomy and the physiological processes of the body in bothhealth and disease. MRI is a form of medical imaging that measures theresponse of the atomic nuclei of body tissues to high-frequency radiowaves when placed in a strong magnetic field, and that produces imagesof the internal organs. MRI scanners, which is based on the science ofnuclear magnetic resonance (NMR), use strong magnetic fields, radiowaves, and field gradients to generate images of the inside of the body.

“Monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological.

“Multivalent binding protein” is used herein to refer to a bindingprotein comprising two or more antigen binding sites (also referred toherein as “antigen binding domains”). A multivalent binding protein ispreferably engineered to have three or more antigen binding sites, andis generally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein that can bind two or morerelated or unrelated targets, including a binding protein capable ofbinding two or more different epitopes of the same target molecule.

“Negative predictive value” or “NPV” as used interchangeably hereinrefers to the probability that a subject has a negative outcome giventhat they have a negative test result.

“Reference level” as used herein refers to an assay cutoff value that isused to assess diagnostic, prognostic, or therapeutic efficacy and thathas been linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.). An“absolute amount” as used herein refers to the absolute value of achange or difference between at least two assay results taken or sampledat different time points and, which similar to a reference level, hasbeen linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.).“Absolute value” as used herein refers to the magnitude of a real number(such as, for example, the difference between two compared levels (suchas levels taken at a first time point and levels taken at a second timepoint)) without regard to its sign, i.e., regardless of whether it ispositive or negative.

This disclosure provides exemplary reference levels and absolute amounts(e.g., calculated by comparing reference levels at different timepoints). However, it is well-known that reference levels and absoluteamounts may vary depending on the nature of the immunoassay (e.g.,antibodies employed, reaction conditions, sample purity, etc.) and thatassays can be compared and standardized. It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific reference levels andabsolute amounts for those other immunoassays based on the descriptionprovided by this disclosure. Whereas the precise value of the referencelevel and absolute amounts may vary between assays, the findings asdescribed herein should be generally applicable and capable of beingextrapolated to other assays.

“Point-of-care device” refers to a device used to provide medicaldiagnostic testing at or near the point-of-care (namely, outside of alaboratory), at the time and place of patient care (such as in ahospital, physician's office, urgent or other medical care facility, apatient's home, a nursing home and/or a long term care and/or hospicefacility). Examples of point-of-care devices include those produced byAbbott Laboratories (Abbott Park, Ill.) (e.g., i-STAT and i-STATAlinity, Universal Biosensors (Rowville, Australia) (see US2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical LabProducts (Los Angeles, USA).

“Positive predictive value” or “PPV” as used interchangeably hereinrefers to the probability that a subject has a positive outcome giventhat they have a positive test result.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a reference level or control level (e.g.,“low”, “medium”, or “high” levels), can be used. Multiple calibrators(i.e., more than one calibrator or a varying amount of calibrator(s))can be used in conjunction to comprise a “sensitivity panel.”

A “receiver operating characteristic” curve or “ROC” curve refers to agraphical plot that illustrates the performance of a binary classifiersystem as its discrimination threshold is varied. For example, an ROCcurve can be a plot of the true positive rate against the false positiverate for the different possible cutoff points of a diagnostic test. Itis created by plotting the fraction of true positives out of thepositives (TPR=true positive rate) vs. the fraction of false positivesout of the negatives (FPR=false positive rate), at various thresholdsettings. TPR is also known as sensitivity, and FPR is one minus thespecificity or true negative rate. The ROC curve demonstrates thetradeoff between sensitivity and specificity (any increase insensitivity will be accompanied by a decrease in specificity); thecloser the curve follows the left-hand border and then the top border ofthe ROC space, the more accurate the test; the closer the curve comes tothe 45-degree diagonal of the ROC space, the less accurate the test; theslope of the tangent line at a cutoff point gives the likelihood ratio(LR) for that value of the test; and the area under the curve is ameasure of test accuracy.

“Recombinant antibody” and “recombinant antibodies” refer to antibodiesprepared by one or more steps, including cloning nucleic acid sequencesencoding all or a part of one or more monoclonal antibodies into anappropriate expression vector by recombinant techniques and subsequentlyexpressing the antibody in an appropriate host cell. The terms include,but are not limited to, recombinantly produced monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multi-specific or multi-valent structures formed fromantibody fragments, bifunctional antibodies, heteroconjugate Abs,dual-variable domain immunoglobulins (DVD-Ig®s), and other antibodies asdescribed in (i) herein. (Dual-variable domain immunoglobulins andmethods for making them are described in Wu, C., et al., NatureBiotechnology, 25:1290-1297 (2007)). The term “bifunctional antibody,”as used herein, refers to an antibody that comprises a first arm havinga specificity for one antigenic site and a second arm having aspecificity for a different antigenic site, i.e., the bifunctionalantibodies have a dual specificity.

“Risk assessment,” “risk classification,” “risk identification,” or“risk stratification” of subjects (e.g., patients) as used herein refersto the evaluation of factors including biomarkers, to predict the riskof occurrence of future events including disease onset or diseaseprogression, so that treatment decisions regarding the subject may bemade on a more informed basis.

“Sample,” “test sample,” “specimen,” “biological sample”, “sample from asubject,” and “patient sample” as used herein may be usedinterchangeable and may be a sample of blood such as whole blood,tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid,placental cells or tissue, endothelial cells, leukocytes, or monocytes.In some embodiments, the sample is a whole blood sample. In someembodiments, the sample is a serum sample. In yet other embodiments, thesample is a plasma sample. The sample can be used directly as obtainedfrom a patient or can be pre-treated, such as by filtration,distillation, extraction, concentration, centrifugation, inactivation ofinterfering components, addition of reagents, and the like, to modifythe character of the sample in some manner as discussed herein orotherwise as is known in the art.

A variety of cell types, tissue, or bodily fluid may be utilized toobtain a sample. Such cell types, tissues, and fluid may includesections of tissues such as biopsy and autopsy samples, frozen sectionstaken for histologic purposes, blood (such as whole blood), plasma,serum, red blood cells, platelets, interstitial fluid, cerebral spinalfluid, etc. Cell types and tissues may also include lymph fluid,cerebrospinal fluid, a fluid collected by A tissue or cell type may beprovided by removing a sample of cells from a human and a non-humananimal, but can also be accomplished by using previously isolated cells(e.g., isolated by another person, at another time, and/or for anotherpurpose). Archival tissues, such as those having treatment or outcomehistory, may also be used. Protein or nucleotide isolation and/orpurification may not be necessary.

“Sensitivity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is positive that are correctly identifiedas positive (e.g., correctly identifying those subjects with a diseaseor medical condition for which they are being tested). For example, thismight include correctly identifying subjects as having a TBI from thosewho do not have a TBI, correctly identifying subjects having a moderate,severe, or moderate to severe TBI from those having a mild TBI,correctly identifying subjects as having a mild TBI from those having amoderate, severe, or moderate to severe TBI, correctly identifyingsubjects as having a moderate, severe, or moderate to severe TBI fromthose having no TBI or correctly identifying subjects as having a mildTBI from those having no TBI, correctly identifying subjects as likelyto benefit from imaging or a head CT scan or a MRI from those who arenot likely to benefit from a head imaging or a CT scan or MRI, etc.).

“Specificity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is negative that are correctly identifiedas negative (e.g., correctly identifying those subjects who do not havea disease or medical condition for which they are being tested). Forexample, this might include correctly identifying subjects having an TBIfrom those who do not have a TBI, correctly identifying subjects nothaving a moderate, severe, or moderate to severe TBI from those having amild TBI, correctly identifying subjects as not having a mild TBI fromthose having a moderate, severe, or moderate to severe TBI or correctlyidentifying subjects as not having any TBI, or correctly identifyingsubjects as having a mild TBI from those having no TBI, etc.).

“Solid phase” or “solid support” as used interchangeably herein, refersto any material that can be used to attach and/or attract and immobilize(1) one or more capture agents or capture specific binding partners, or(2) one or more detection agents or detection specific binding partners.The solid phase can be chosen for its intrinsic ability to attract andimmobilize a capture agent. Alternatively, the solid phase can haveaffixed thereto a linking agent that has the ability to attract andimmobilize the (1) capture agent or capture specific binding partner, or(2) detection agent or detection specific binding partner. For example,the linking agent can include a charged substance that is oppositelycharged with respect to the capture agent (e.g., capture specificbinding partner) or detection agent (e.g., detection specific bindingpartner) itself or to a charged substance conjugated to the (1) captureagent or capture specific binding partner or (2) detection agent ordetection specific binding partner. In general, the linking agent can beany binding partner (preferably specific) that is immobilized on(attached to) the solid phase and that has the ability to immobilize the(1) capture agent or capture specific binding partner, or (2) detectionagent or detection specific binding partner through a binding reaction.The linking agent enables the indirect binding of the capture agent to asolid phase material before the performance of the assay or during theperformance of the assay. For examples, the solid phase can be plastic,derivatized plastic, magnetic, or non-magnetic metal, glass or silicon,including, for example, a test tube, microtiter well, sheet, bead,microparticle, chip, and other configurations known to those of ordinaryskill in the art.

“Specific binding” or “specifically binding” as used herein may refer tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, wherein the interaction is dependent upon the presenceof a particular structure (e.g., an antigenic determinant or epitope) onthe chemical species; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzymes and enzyme inhibitors, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes and fragments thereof, whether isolatedor recombinantly produced.

“Statistically significant” as used herein refers to the likelihood thata relationship between two or more variables is caused by somethingother than random chance. Statistical hypothesis testing is used todetermine whether the result of a data set is statistically significant.In statistical hypothesis testing, a statistical significant result isattained whenever the observed p-value of a test statistic is less thanthe significance level defined of the study. The p-value is theprobability of obtaining results at least as extreme as those observed,given that the null hypothesis is true. Examples of statisticalhypothesis analysis include Wilcoxon signed-rank test, t-test,Chi-Square or Fisher's exact test. “Significant” as used herein refersto a change that has not been determined to be statistically significant(e.g., it may not have been subject to statistical hypothesis testing).

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. In someembodiments, the subject is a human. The subject or patient may beundergoing other forms of treatment. In some embodiments, when thesubject is a human, the subject does not include any humans who havesuffered a cerebrovascular accident (e.g., a stroke).

“Treat,” “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease and/or injury, or one or more symptoms of such disease, to whichsuch term applies. Depending on the condition of the subject, the termalso refers to preventing a disease, and includes preventing the onsetof a disease, or preventing the symptoms associated with a disease. Atreatment may be either performed in an acute or chronic way. The termalso refers to reducing the severity of a disease or symptoms associatedwith such disease prior to affliction with the disease. Such preventionor reduction of the severity of a disease prior to affliction refers toadministration of a pharmaceutical composition to a subject that is notat the time of administration afflicted with the disease. “Preventing”also refers to preventing the recurrence of a disease or of one or moresymptoms associated with such disease. “Treatment” and“therapeutically,” refer to the act of treating, as “treating” isdefined above.

“Traumatic Brain Injury” or “TBI” as used interchangeably herein refersto a complex injury with a broad spectrum of symptoms and disabilities.TBI is most often an acute event similar to other injuries. TBI can beclassified as “mild,” “moderate,” or “severe.” The causes of TBI arediverse and include, for example, physical shaking by a person, a caraccident, injuries from firearms, cerebral vascular accidents (e.g.,strokes), falls, explosions or blasts and other types of blunt forcetrauma. Other causes of TBI include the ingestion and/or exposure to oneor more chemicals or toxins (such as fires, molds, asbestos, pesticidesand insecticides, organic solvents, paints, glues, gases (such as carbonmonoxide, hydrogen sulfide, and cyanide), organic metals (such as methylmercury, tetraethyl lead and organic tin), one or more drugs of abuse orcombinations thereof). Alternatively, TBI can occur in subjectssuffering from an autoimmune disease, a metabolic disorder, a braintumor, hypoxia, one or more viruses, meningitis, hydrocephalus orcombinations thereof. Young adults and the elderly are the age groups athighest risk for TBI. In certain embodiments herein, traumatic braininjury or TBI does not include and specifically excludes cerebralvascular accidents such as strokes.

“Mild TBI” as used herein refers to a brain injury where loss ofconsciousness is brief and usually a few seconds or minutes and/orconfusion and disorientation is shorter than 1 hour. Mild TBI is alsoreferred to as a concussion, minor head trauma, minor TBI, minor braininjury, and minor head injury. While MRI and CT scans are often normal,the individual with mild TBI may have cognitive problems such asheadache, difficulty thinking, memory problems, attention deficits, moodswings and frustration.

Mild TBI is the most prevalent TBI and is often missed at time ofinitial injury. Typically, a subject has a Glasgow Coma scale number ofbetween 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of peoplewith mild TBI have symptoms that last 3 months or more. Mild TBI isdefined as the result of the forceful motion of the head or impactcausing a brief change in mental status (confusion, disorientation orloss of memory) or loss of consciousness for less than 30 minutes.Common symptoms of mild TBI include fatigue, headaches, visualdisturbances, memory loss, poor attention/concentration, sleepdisturbances, dizziness/loss of balance, irritability-emotionaldisturbances, feelings of depression, and seizures. Other symptomsassociated with mild TBI include nausea, loss of smell, sensitivity tolight and sounds, mood changes, getting lost or confused, and/orslowness in thinking.

“Moderate TBI” as used herein refers to a brain injury where loss ofconsciousness and/or confusion and disorientation is between 1 and 24hours and the subject has a Glasgow Coma scale number of between 9-13(such as 9-12 or 9-13). The individual with moderate TBI have abnormalbrain imaging results. “Severe TBI” as used herein refers to a braininjury where loss of consciousness is more than 24 hours and memory lossafter the injury or penetrating skull injury longer than 24 hours andthe subject has a Glasgow Coma scale number between 3-8. The deficitsrange from impairment of higher level cognitive functions to comatosestates. Survivors may have limited function of arms or legs, abnormalspeech or language, loss of thinking ability or emotional problems.Individuals with severe injuries can be left in long-term unresponsivestates. For many people with severe TBI, long-term rehabilitation isoften necessary to maximize function and independence.

“Moderate to severe” TBI as used herein refers to a spectrum of braininjury that includes moderate to severe and thus encompasses moderateTBI alone, severe TBI alone and moderate to severe TBI combined.Subjects suffering from a moderate to severe TBI can have a Glasgow Comascale number of between 3-13 (such as 3-12 or 3-13). For example, insome clinical situations, a subject may initially be diagnosed as havinga moderate TBI but who, over the course of time (minutes, hours ordays), progress to having a severe TBI (such, as for example, insituations when there is a brain bleed). Such subjects would be examplesof patients that could be classified as “moderate to severe”. Commonsymptoms of moderate to severe TBI include cognitive deficits includingdifficulties with attention, concentration, distractibility, memory,speed of processing, confusion, preservation, impulsiveness, languageprocessing, and/or “executive functions”, not understanding the spokenword (receptive aphasia), difficulty speaking and being understood(expressive aphasia), slurred speech, speaking very fast or very slow,problems reading, problems writing, difficulties with interpretation oftouch, temperature, movement, limb position and fine discrimination, theintegration or patterning of sensory impressions into psychologicallymeaningful data, partial or total loss of vision, weakness of eyemuscles and double vision (diplopia), blurred vision, problems judgingdistance, involuntary eye movements (nystagmus), intolerance of light(photophobia), hearing, such as decrease or loss of hearing, ringing inthe ears (tinnitus), increased sensitivity to sounds, loss or diminishedsense of smell (anosmia), loss or diminished sense of taste, theconvulsions associated with epilepsy that can be several types and caninvolve disruption in consciousness, sensory perception, or motormovements, control of bowel and bladder, sleep disorders, loss ofstamina, appetite changes, regulation of body temperature, menstrualdifficulties, dependent behaviors, emotional ability, lack ofmotivation, irritability, aggression, depression, disinhibition, ordenial/lack of awareness.

“Variant” is used herein to describe a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Representative examples of “biological activity”include the ability to be bound by a specific antibody or to promote animmune response. Variant is also used herein to describe a protein withan amino acid sequence that is substantially identical to a referencedprotein with an amino acid sequence that retains at least one biologicalactivity. A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity, degree, and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to refer to an antigenically reactivefragment of an anti-cTnI antibody that differs from the correspondingfragment of anti-cTnI antibody in amino acid sequence but is stillantigenically reactive and can compete with the corresponding fragmentof anti-cTnI antibody for binding with cTnI. “Variant” also can be usedto describe a polypeptide or a fragment thereof that has beendifferentially processed, such as by proteolysis, phosphorylation, orother post-translational modification, yet retains its antigenreactivity.

“Vector” is used herein to describe a nucleic acid molecule that cantransport another nucleic acid to which it has been linked. One type ofvector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors can replicate autonomously in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”). Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. “Plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions, can be used. In this regard,RNA versions of vectors (including RNA viral vectors) may also find usein the context of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. For example,any nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those that are well known and commonly used in the art. Themeaning and scope of the terms should be clear; in the event, however ofany latent ambiguity, definitions provided herein take precedent overany dictionary or extrinsic definition. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

2. Methods of Aiding in the Diagnosis and Evaluation of Whether a HumanSubject has Sustained or may have Sustained an (or has an actual orsuspected) Injury to the Head Using Cardiac Troponin I (cTnI)

The present disclosure relates, among other methods, to a method ofaiding in the diagnosis and evaluation of whether a human subject hassustained or may have sustained (or has an actual or suspected) aninjury to the head using cardiac troponin I (cTnI) levels or changes incTnI levels. Specifically, the methods described herein can aid indetermining the extent of traumatic brain injury in a human subject withan actual or suspected injury to the head, e.g., determining whether thesubject has mild traumatic brain injury or moderate, severe, or moderateto severe traumatic brain injury. As used here, “determining whether thesubject has mild traumatic brain injury or moderate, severe, or moderateto severe traumatic brain injury” refers to the fact that theaforementioned method can be used, e.g., with other information (e.g.,clinical assessment data), to determine that the subject is more likelythan not to have mild traumatic brain injury, moderate, severe, ormoderate to severe traumatic brain injury or no traumatic brain injury.The method can include performing an assay on a sample obtained from thehuman subject within about 24 hours, such as within about 2 hours, afteran actual or suspected injury to the head to measure or detect a levelof cardiac troponin I (cTnI) in the sample and determining whether thesubject has sustained a mild, a moderate, severe, moderate to severetraumatic brain injury (TBI) or no TBI. In some embodiments, the subjectis determined as having (1) a moderate, severe, or moderate to severeTBI when the level of cTnI in the sample is higher than a referencelevel of cTnI, or (2) a mild TBI when the level of cTnI in the sample islower than a reference level of cTnI. The sample can be a biologicalsample. In some aspects, the biological sample is a whole blood sample.In other aspects, the biological sample is a serum sample. In yet otheraspects, the biological sample is a plasma sample.

In some embodiments, the method can include obtaining a sample withinabout 24 hours, such as within about 2 hours, of an actual or suspectedinjury to the subject and contacting the sample with an antibody forcTnI to allow formation of a complex of the antibody and cTnI. Themethod also includes detecting the resulting antibody-cTnI complex.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of a suspect injury to the head.

In some embodiments, the sample is taken from the human subject withinabout 2 hours of (an actual) injury or suspected injury to the head. Forexample, the sample can be taken from the human subject within about 0minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about2 hours of injury or suspected injury to the head. In some embodiments,the onset of the presence of cTnI appears within about 0 minutes, about1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 60 minutes, about 90 minutes, or about 2 hours afterinjury to the head.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cardiac troponin is determined at oneor more time points. In certain embodiments, the subject may besuspected of having a mild traumatic brain injury based on the GlasgowComa Scale score. In certain embodiments, the subject may be suspectedof having a mild traumatic brain injury based on an abnormal head CT. Insome embodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the reference level of cTnI is correlated withsubjects having a moderate, severe, or moderate to severe traumaticbrain injury. In some embodiments, the reference level of cTnI iscorrelated with a Glasgow Coma Scale score of 3-12 (moderate to severeTBI). In some embodiments, the reference level of cTnI is correlatedwith a Glasgow Coma Scale score of 3-8 (a severe TBI). In someembodiments, the reference level of cTnI is correlated with a GlasgowComa Scale score of 9-13 (a moderate TBI). In some embodiments, thesubject is suspected as having mild traumatic brain injury based on theGlasgow Coma Scale score. In some embodiments, the reference level ofcTnI is correlated with subjects having mild traumatic brain injury. Insome embodiments, the reference level of cTnI is correlated with aGlasgow Coma Scale score of 13-15 (a mild TBI).

Generally, a reference level of cTnI can also be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In certain embodiments, the reference level may be correlated withcontrol subjects that have not sustained a head injury.

In some embodiments, the reference level of cTnI is determined by anassay having a sensitivity of between at least about 65% to about 100%and a specificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 65% to about100%, between at least about 65% to at least about 99%, between at leastabout 65% to at least about 95%, between at least about 65% to at leastabout 90%, between at least about 65% to at least about 85%, between atleast about 65% to at least about 80%, between at least about 65% to atleast about 75%, between at least about 65% to at least about 70%,between at least about 75% to about 100%, between at least about 75% toat least about 99%, between at least about 75% to at least about 95%,between at least about 75% to at least about 90%, between at least about75% to at least about 85%, between at least about 75% to at least about80%, between at least about 85% to about 100%, between at least about85% to at least about 99%, between at least about 85% to at least about95%, between at least about 85% to at least about 90%, between at leastabout 95% to about 100%, or between at least about 95% to at least about99%. In some embodiments, the sensitivity is at least about 65.0%, atleast about 70.0%, at least about 75.0%, at least about 80.0%, at leastabout 85.0%, at least about 87.5%, at least about 90.0%, at least about95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%,at least about 99.3%, at least about 99.4%, at least about 99.5%, atleast about 99.6%, at least about 99.7%, at least about 99.8%, at leastabout 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the amount of cardiac troponin I in the sample isfrom about 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about 45 pg/mL,about 1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35 pg/mL, about1 pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL, about 1pg/mL to about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about 1 pg/mLto about 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mL toabout 8 pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about 6pg/mL, about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL,about 1 pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1pg/mL to about 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5pg/mL to about 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5pg/mL to about 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5pg/mL to about 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5pg/mL to about 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5pg/mL to about 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5pg/mL to about 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5pg/mL to about 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5pg/mL to about 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mLto about 50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL toabout 40 pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about30 pg/mL, about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20pg/mL, about 2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL,about 2 pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2pg/mL to about 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL toabout 5 pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL,about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mLto about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL toabout 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL,about 4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about4 pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4pg/mL to about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mLto about 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL toabout 10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5pg/mL to about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mLto about 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL toabout 25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about15 pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9pg/mL, about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL,about 5 pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6pg/mL to about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mLto about 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL toabout 25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about15 pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9pg/mL, about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL,about 7 pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about7 pg/mL to about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7pg/mL to about 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mLto about 20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL toabout 10 pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL,about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mLto about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL toabout 50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about40 pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30pg/mL, about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL,about 9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about10 pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the amount of cTnI can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, or atleast about 50 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject assessed as having a moderate, severe, or a moderate to severetraumatic brain injury with a traumatic brain injury treatment, asdescribed below. In some embodiments, the method further includesmonitoring the human subject assessed as having mild traumatic braininjury, as described below. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS). Also, the assay can be employed in a clinical chemistry formatsuch as would be known by one of ordinary skill in the art. Such assaysare described in further detail herein in Sections 11-13. It is known inthe art that the values (e.g., reference levels, cutoffs, thresholds,specificities, sensitivities, concentrations of calibrators and/orcontrols etc.) used in an assay that employs specific sample type (e.g.,such as an immunoassay that utilizes serum or a point-of-care devicethat employs whole blood) can be extrapolated to other assay formatsusing known techniques in the art, such as assay standardization. Forexample, one way in which assay standardization can be performed is byapplying a factor to the calibrator employed in the assay to make thesample concentration read higher or lower to get a slope that alignswith the comparator method. Other methods of standardizing resultsobtained on one assay to another assay are well known and have beendescribed in the literature (See, for example, David Wild, ImmunoassayHandbook, 4^(th) edition, chapter 3.5, pages 315-322, the contents ofwhich are herein incorporated by reference).

3. Method of Aiding in the Determination of Whether to Perform a CT Scanon a Human Subject Who May Have Sustained or has Sustained an (or has anActual or Suspected) Injury to the Head Using Cardiac Troponin I (cTnI)

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform a computerized tomography (CT)scan on a human subject who has sustained or may have sustained an (orhas an actual or suspected) injury to the head. As used here,“determination of whether to perform a CT scan on a human subject”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have a positive head CTscan. Specifically, such a method can comprise the steps of: (a)performing an assay on a sample obtained from the subject within about24 hours, such as within about 2 hours, after an actual or suspectedinjury to the head to measure or detect a level of cardiac troponin I(cTnI) in the sample; and (b) performing a CT scan on the subject whenthe level of cTnI in the sample is higher than a reference level of cTnIand not performing a CT scan on the subject when the level of cTnI inthe sample is lower than a reference level of cTnI. The sample can be abiological sample.

In some embodiments, the method can include obtaining a sample withinabout 24 hours, such as within about 2 hours, of an actual or suspectedinjury to the subject and contacting the sample with an antibody forcTnI to allow formation of a complex of the antibody and cTnI. Themethod also includes detecting the resulting antibody-cTnI complex.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of an actual or suspect injury to the head.

In some embodiments, the sample is taken from the human subject withinabout 2 hours of injury or suspected injury to the head. For example,the sample can be taken from the human subject within about 0 minutes,about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 60 minutes, about 90 minutes, or about 2 hours of injuryor suspected injury to the head. In some embodiments, the onset of thepresence of cTnI appears within about 0 minutes, about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60minutes, about 90 minutes, or about 2 hours after injury to the head.

In some embodiments, the subject has received a CT scan before or afterthe assay is performed. In some embodiments, the subject is suspected ashaving a traumatic brain injury based on the CT scan. In someembodiments, the reference level of cTnI is correlated with positivehead CT scan.

Generally, a reference level of cTnI can be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In some embodiments, the reference level of cTnI is determined by anassay having a sensitivity of between at least about 65% to about 100%and a specificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 65% to about100%, between at least about 65% to at least about 99%, between at leastabout 65% to at least about 95%, between at least about 65% to at leastabout 90%, between at least about 65% to at least about 85%, between atleast about 65% to at least about 80%, between at least about 65% to atleast about 75%, between at least about 65% to at least about 70%,between at least about 75% to about 100%, between at least about 75% toat least about 99%, between at least about 75% to at least about 95%,between at least about 75% to at least about 90%, between at least about75% to at least about 85%, between at least about 75% to at least about80%, between at least about 85% to about 100%, between at least about85% to at least about 99%, between at least about 85% to at least about95%, between at least about 85% to at least about 90%, between at leastabout 95% to about 100%, or between at least about 95% to at least about99%. In some embodiments, the sensitivity is at least about 65.0%, atleast about 70.0%, at least about 75.0%, at least about 80.0%, at leastabout 85.0%, at least about 87.5%, at least about 90.0%, at least about95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%,at least about 99.3%, at least about 99.4%, at least about 99.5%, atleast about 99.6%, at least about 99.7%, at least about 99.8%, at leastabout 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the amount of cardiac troponin I in the sample isfrom about 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about 45 pg/mL,about 1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35 pg/mL, about1 pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL, about 1pg/mL to about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about 1 pg/mLto about 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mL toabout 8 pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about 6pg/mL, about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL,about 1 pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1pg/mL to about 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5pg/mL to about 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5pg/mL to about 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5pg/mL to about 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5pg/mL to about 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5pg/mL to about 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5pg/mL to about 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5pg/mL to about 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5pg/mL to about 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mLto about 50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL toabout 40 pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about30 pg/mL, about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20pg/mL, about 2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL,about 2 pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2pg/mL to about 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL toabout 5 pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL,about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mLto about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL toabout 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL,about 4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about4 pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4pg/mL to about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mLto about 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL toabout 10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5pg/mL to about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mLto about 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL toabout 25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about15 pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9pg/mL, about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL,about 5 pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6pg/mL to about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mLto about 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL toabout 25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about15 pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9pg/mL, about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL,about 7 pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about7 pg/mL to about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7pg/mL to about 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mLto about 20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL toabout 10 pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL,about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mLto about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL toabout 50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about40 pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30pg/mL, about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL,about 9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about10 pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the amount of cTnI can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, or atleast about 50 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject with a traumatic brain injury treatment and/or monitoring thehuman subject, as described below. In some embodiments, the methodfurther includes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 11-13.

4. Methods of Aiding in the Diagnosis and Evaluation of Whether a HumanSubject may have or has Sustained an (or has an Actual or Suspected)Injury to the Head based on Changes in Cardiac Troponin I (cTnI) Levels

The present disclosure relates, among other methods, to a method ofaiding in the diagnosis and evaluation of whether a human subject hassustained or may have sustained an (or has an actual or suspected)injury to the head. The method can aid in determining the extent oftraumatic brain injury in a human subject with an actual or suspectedinjury to the head, e.g., determining whether the subject has mildtraumatic brain injury or a moderate, severe, or moderate to severetraumatic brain injury. As used here, “determining whether the subjecthas mild traumatic brain injury or moderate, severe, or moderate tosevere traumatic brain injury” refers to the fact that theaforementioned method can be used, e.g., with other information (e.g.,clinical assessment data), to determine that the subject is more likelythan not to have mild traumatic brain injury or moderate, severe, ormoderate to severe traumatic brain injury. The method can includeperforming an assay on at least two samples obtained from the subject,the first sample taken from the subject within about 24 hours, such aswithin about 2 hours, after an injury or suspected injury to the headand the second sample taken from the subject from about 3 to about 6hours after the first sample is taken; detecting in the at least twosamples cardiac troponin I (cTnI); and determining whether the subjecthas sustained a mild or a moderate, severe, or moderate to severetraumatic brain injury (TBI). The subject is determined as having (1) amoderate, severe, or moderate to severe traumatic brain injury when thelevel of cTnI decreases or increases by at least an absolute amount fromthe first sample to the second sample or (2) a mild traumatic braininjury when there is no decrease or increase by at least an absoluteamount in the level of cTnI from the first sample to the second sample.The samples can be biological samples.

In an alternative, the method can include performing an assay on atleast two samples obtained from the subject, the first sample taken fromthe subject within about 24 hours, such as within about 2 hours, afteran injury or suspected injury to the head and the second sample takenfrom the subject from about 3 to about 6 hours after the first sample istaken; detecting in the at least two samples cTnI; and determiningwhether the subject has sustained a mild or a moderate, severe, ormoderate to severe traumatic brain injury (TBI), wherein the subject isdetermined as having (1) a moderate, severe, or a moderate to severetraumatic brain injury when the level of cTnI decreases or increases byat least a first absolute amount from the first sample to the secondsample or (2) a mild traumatic brain injury when there is no decrease orincrease by at least a second absolute amount in the level of cTnI fromthe first sample to the second sample. The samples can be biologicalsamples.

In some embodiments, the method can include contacting the samples withan antibody for cTnI, to allow formation of a complex of the antibodyand cTnI. The method also includes detecting the resulting antibody-cTnIcomplex to determine the levels of cTnI for each of the first sample andsecond sample. The onset of the presence of cTnI appears within about 0to about 2 hours after the onset of the suspected injury. In someembodiments, the onset of the presence of cTnI appears within about 0minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about2 hours after injury to the head.

In some embodiments, the first sample is obtained at a first time pointwithin about 24 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 24 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample may beobtained or taken from the subject within about 0 minutes, within about1 minute, within about 2 minutes, within about 3 minutes, within about 4minutes, within about 5 minutes, within about 6 minutes, within about 7minutes, within about 8 minutes, within about 9 minutes, within about 10minutes, within about 11 minutes, within about 12 minutes, within about13 minutes, within about 14 minutes, within about 15 minutes, withinabout 20 minutes, within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours of an injury or suspected injury to thehead.

In some embodiments, the first sample is obtained at a first time pointwithin about 2 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 2 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample is takenabout 0 to about 2 hours after the injury or suspected injury to thehead. For example, the first sample can be taken between about 0 toabout 2 hours, about 0 hours to about 90 minutes, about 0 hours to about60 minutes, about 0 hours to about 45 minutes, about 0 hours to about 30minutes, about 0 hours to about 20 minutes, about 0 hours to about 15minutes, about 0 hours to about 10 minutes, about 0 hours to about 5minutes, about 5 minutes to about 90 minutes, about 5 minutes to about60 minutes, about 5 minutes to about 45 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 20 minutes, about 5 minutesto about 15 minutes, about 5 minutes to about 10 minutes, about 10minutes to about 90 minutes, about 10 minutes to about 60 minutes, about10 minutes to about 45 minutes, about 10 minutes to about 30 minutes,about 10 minutes to about 20 minutes, about 10 minutes to about 15minutes, about 15 minutes to about 90 minutes, about 15 minutes to about60 minutes, about 15 minutes to about 45 minutes, about 15 minutes toabout 30 minutes, about 15 minutes to about 20 minutes, about 20 minutesto about 90 minutes, about 20 minutes to about 60 minutes, about 20minutes to about 45 minutes, or about 20 minutes to about 30 minutesafter the suspected injury. For example, the first sample can be takenfrom the human subject within about 0 minutes, about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60minutes, about 90 minutes, or about 2 hours of injury or suspectedinjury to the head.

In some embodiments, the second sample is taken about 1 hour to about 10hours after the first time point, such as about 3 hours to about 6 hoursafter the first time point. In some embodiments, the second sample istaken about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, orabout 10 hours after the first sample.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cardiac troponin is determined at oneor more time points. In certain embodiments, the subject may besuspected of having a mild traumatic brain injury based on the GlasgowComa Scale score. In certain embodiments, the subject may be suspectedof having a mild traumatic brain injury based on an abnormal head CT. Insome embodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the reference level of cTnI is correlated withsubjects having a moderate, severe, or moderate to severe traumaticbrain injury. In some embodiments, the reference level of cTnI iscorrelated with a Glasgow Coma Scale score of 3-12 (moderate to severeTBI). In some embodiments, the reference level of cTnI is correlatedwith a Glasgow Coma Scale score of 3-8 (a severe TBI). In someembodiments, the reference level of cTnI is correlated with a GlasgowComa Scale score of 9-13 (a moderate TBI). In some embodiments, thesubject is suspected as having mild traumatic brain injury based on theGlasgow Coma Scale score. In some embodiments, the reference level ofcTnI is correlated with subjects having mild traumatic brain injury. Insome embodiments, the reference level of cTnI is correlated with aGlasgow Coma Scale score of 13-15 (a mild TBI).

In some embodiments, the absolute amount can be determined by an assayhaving a sensitivity of between at least about 65% to about 100% and aspecificity of between at least about 65% to about 100%. For example,the absolute amount can be determined by an assay having a sensitivityof between at least about 80% to 100% and a specificity of between atleast about 65% to 100%. In some embodiments, the sensitivity is atleast about 65.0%, the sensitivity is at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%. In someembodiments, the specificity is at least about 65.0%, at least about70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%,at least about 90.0%, at least about 91.0%, at least about 92.0%, atleast about 93. %, at least about 94.0%, at least about 95.0%, at leastabout 96.0%, at least about 97.0%, at least about 98.0%, at least about99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%,at least about 99.4%, at least about 99.5%, at least about 99.6%, atleast about 99.7%, at least about 99.8%, at least about 99.9%, or atleast about 100.0%. For example, the sensitivity is at least about 100%and the specificity is at least about 75%, the sensitivity is at leastabout 99% and the specificity is at least about 99%, or the sensitivityis at least about 87% and the specificity is at least about 95%.

In some embodiments, the absolute amount of cardiac troponin I in thesample is from about 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about45 pg/mL, about 1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35pg/mL, about 1 pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL,about 1 pg/mL to about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about1 pg/mL to about 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mLto about 8 pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about6 pg/mL, about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL,about 1 pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1pg/mL to about 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5pg/mL to about 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5pg/mL to about 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5pg/mL to about 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5pg/mL to about 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5pg/mL to about 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5pg/mL to about 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5pg/mL to about 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5pg/mL to about 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mLto about 50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL toabout 40 pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about30 pg/mL, about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20pg/mL, about 2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL,about 2 pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2pg/mL to about 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL toabout 5 pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL,about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mLto about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL toabout 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL,about 4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about4 pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4pg/mL to about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mLto about 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL toabout 10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5pg/mL to about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mLto about 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL toabout 25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about15 pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9pg/mL, about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL,about 5 pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6pg/mL to about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mLto about 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL toabout 25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about15 pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9pg/mL, about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL,about 7 pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about7 pg/mL to about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7pg/mL to about 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mLto about 20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL toabout 10 pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL,about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mLto about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL toabout 50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about40 pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30pg/mL, about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL,about 9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about10 pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the absolute amount can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, or atleast about 50 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury with a traumatic brain injury treatment, asdescribed below. In some embodiments, the method further includesmonitoring the human subject assessed as having mild traumatic braininjury, as described below. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical, and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 11-13.

5. Method of Aiding in the Determination of Whether to Perform a CT Scanon a Human Subject Who may have Sustained or Sustained (or has an Actualor Suspected) an Injury to the Head based on Changes in Cardiac TroponinI (cTnI) Levels

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform a computerized tomography (CT)scan on a human subject who has sustained or may have sustained an (orhas an actual or suspected) injury to the head. As used here,“determination of whether to perform a CT scan on a human subject”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have a positive head CTscan. Specifically, such a method can comprise the steps of: performingan assay on at least two samples obtained from the subject, the firstsample taken from the subject within about 24 hours, such as withinabout 2 hours, of the suspected injury and the second sample taken fromthe subject from about 3 to about 6 hours after the first sample istaken; detecting in the at least two samples cardiac troponin I (cTnI);and performing a CT scan on the subject when the level of cTnI decreasesor increases by at least an absolute amount from the first sample to thesecond sample and not performing a CT scan on the subject when there isno decrease or increase by at least an absolute amount in the level ofcTnI from the first sample to the second sample. The samples can bebiological samples.

In some embodiments, the method can include contacting the samples withan antibody for cTnI, to allow formation of a complex of the antibodyand cTnI. The method also includes detecting the resulting antibody-cTnIcomplex to determine the levels of cTnI for each of the first sample andsecond sample. The onset of the presence of cTnI appears within about 0to about 24 hours, such as within about 2 hours, after the onset of thesuspected injury. In some embodiments, the onset of the presence of cTnIappears within about 0 minutes, about 1 minute, about 2 minutes, about 3minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90minutes, or about 2 hours after injury to the head.

In some embodiments, the first sample is obtained at a first time pointwithin about 24 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 24 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample may beobtained or taken from the subject within about 0 minutes, within about1 minute, within about 2 minutes, within about 3 minutes, within about 4minutes, within about 5 minutes, within about 6 minutes, within about 7minutes, within about 8 minutes, within about 9 minutes, within about 10minutes, within about 11 minutes, within about 12 minutes, within about13 minutes, within about 14 minutes, within about 15 minutes, withinabout 20 minutes, within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours of an actual or suspected injury to thehead.

In some embodiments, a first sample is obtained at a first time pointwithin about 2 hours of the suspected injury and a second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point to determine whether thesubject will have a positive or negative head CT scan. In someembodiments, the first sample is taken within about 2 hours after thesuspected injury and the second sample is taken within about 3 hours toabout 6 hours after the first sample. In some embodiments, the firsttime point is about 0 to about 2 hours after the injury or suspectedinjury to the head. For example, the first time point can be betweenabout 0 to about 2 hours, about 0 hours to about 90 minutes, about 0hours to about 60 minutes, about 0 hours to about 45 minutes, about 0hours to about 30 minutes, about 0 hours to about 20 minutes, about 0hours to about 15 minutes, about 0 hours to about 10 minutes, about 0hours to about 5 minutes, about 5 minutes to about 90 minutes, about 5minutes to about 60 minutes, about 5 minutes to about 45 minutes, about5 minutes to about 30 minutes, about 5 minutes to about 20 minutes,about 5 minutes to about 15 minutes, about 5 minutes to about 10minutes, about 10 minutes to about 90 minutes, about 10 minutes to about60 minutes, about 10 minutes to about 45 minutes, about 10 minutes toabout 30 minutes, about 10 minutes to about 20 minutes, about 10 minutesto about 15 minutes, about 15 minutes to about 90 minutes, about 15minutes to about 60 minutes, about 15 minutes to about 45 minutes, about15 minutes to about 30 minutes, about 15 minutes to about 20 minutes,about 20 minutes to about 90 minutes, about 20 minutes to about 60minutes, about 20 minutes to about 45 minutes, or about 20 minutes toabout 30 minutes after the suspected injury.

In some embodiments, the second time point, or optionally a third timepoint or fourth time point, is about 1 hour to about 10 hours after thefirst time point, such as about 3 hours to about 6 hours after the firsttime point. In some embodiments, the second time point is about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hoursafter the first time point.

In some embodiments, the absolute amount can be determined by an assayhaving a sensitivity of between at least about 65% to about 100% and aspecificity of between at least about 65% to about 100%. For example,the absolute amount can be determined by an assay having a sensitivityof between at least about 80% to 100% and a specificity of between atleast about 65% to 100%. In some embodiments, the sensitivity is atleast about 65.0%, the sensitivity is at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%. In someembodiments, the specificity is at least about 65.0%, at least about70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%,at least about 90.0%, at least about 91.0%, at least about 92.0%, atleast about 93. %, at least about 94.0%, at least about 95.0%, at leastabout 96.0%, at least about 97.0%, at least about 98.0%, at least about99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%,at least about 99.4%, at least about 99.5%, at least about 99.6%, atleast about 99.7%, at least about 99.8%, at least about 99.9%, or atleast about 100.0%. For example, the sensitivity is at least about 100%and the specificity is at least about 75%, the sensitivity is at leastabout 99% and the specificity is at least about 99%, or the sensitivityis at least about 87% and the specificity is at least about 95%.

In some embodiments, the absolute amount of cardiac troponin I in thesample is from about 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about45 pg/mL, about 1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35pg/mL, about 1 pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL,about 1 pg/mL to about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about1 pg/mL to about 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mLto about 8 pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about6 pg/mL, about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL,about 1 pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1pg/mL to about 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5pg/mL to about 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5pg/mL to about 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5pg/mL to about 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5pg/mL to about 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5pg/mL to about 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5pg/mL to about 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5pg/mL to about 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5pg/mL to about 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mLto about 50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL toabout 40 pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about30 pg/mL, about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20pg/mL, about 2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL,about 2 pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2pg/mL to about 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL toabout 5 pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL,about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mLto about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL toabout 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL,about 4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about4 pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4pg/mL to about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mLto about 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL toabout 10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5pg/mL to about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mLto about 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL toabout 25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about15 pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9pg/mL, about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL,about 5 pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6pg/mL to about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mLto about 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL toabout 25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about15 pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9pg/mL, about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL,about 7 pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about7 pg/mL to about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7pg/mL to about 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mLto about 20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL toabout 10 pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL,about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mLto about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL toabout 50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about40 pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30pg/mL, about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL,about 9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about10 pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the absolute amount can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, or atleast about 50 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject who was determined to have a CT scan with a traumatic braininjury treatment, as described below. In some embodiments, the methodfurther includes monitoring, as described below, the human subject whowas determined to have a CT scan. In some embodiments, the methodfurther includes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 11-13.

6. Methods of Aiding in Predicting or Predicting the Outcome of a HumanSubject having Mild Traumatic Brain Injury Using Cardiac Troponin I(cTnI)

The present disclosure relates, among other methods, to a method ofaiding in predicting (or predicting) the outcome of a human subjecthaving mild traumatic brain injury (TBI), e.g., determining whether thesubject will have an unfavorable outcome or a favorable outcome. As usedherein, the phrase “determining whether the subject has a favorableoutcome” refers to the fact that the aforementioned method can be used,e.g., with other information (e.g., clinical assessment data), todetermine that the subject is more likely than not to have a positiveoutcome from the mild TBI. Additionally, as used herein, the phrase“determining whether the subject has an unfavorable outcome” refers tothe fact that the aforementioned method can be used, e.g., with otherinformation (e.g., clinical assessment data), to determine that thesubject is more likely than not to have an unfavorable or negativeoutcome from the mild TBI. As mentioned above, the methods describedherein can be used to determine whether a subject diagnosed with a mildTBI is more likely than not to have (1) a favorable outcome (optionally,the favorable outcome can be that the subject fully recovers and doesnot continue to experience one or more symptoms of a mild TBI); or (2)an unfavorable outcome (optionally, the unfavorable outcome can be thatthe subject does not fully recover and does continue to experience oneor more symptoms of a mild TBI).

Alternatively and optionally, a favorable outcome can mean that thesubject is more likely than not to suffer no more than onepost-concussion syndrome symptom as a result of the mild TBI such as:(a) physical difficulties (e.g., headaches, dizziness, fatigue,sensitivity to light noise and light, etc.); (b) cognitive difficulties(e.g., trouble concentration, memory problems, restlessness, etc.); (c)emotional difficulties (e.g., personality changes, irritability,depression, apathy, etc.); or (d) sleep difficulties (e.g., insomnia,etc.). Alternatively and option, subject who have an unfavorable outcomeare more likely to suffer from more than one post-concussion syndromesymptom such as: (a) physical difficulties (e.g., headaches, dizziness,fatigue, sensitivity to light noise and light, etc.); (b) cognitivedifficulties (e.g., trouble concentration, memory problems,restlessness, etc.); (c) emotional difficulties (e.g, personalitychanges, irritability, depression, apathy, etc.); (d) sleep difficulties(e.g., insomnia, etc.); or (e) any combinations of (a)-(d)).Alternatively and optionally, an unfavorable outcome can also mean thata subject exhibits one or more symptoms of mild TBI. Alternatively andoptionally, an unfavorable outcome can also mean that the subject'sconditions worsens from mild TBI to moderate, moderate to severe orsevere. Additionally, subjects having a favorable outcome are likely tohave a GOSE score of 5 or greater whereas subjects having an unfavorableoutcome are likely to have a GOSE score of less than 5.

At the time of the present disclosure it was known in the art that cTnIlevels are elevated in subjects following severe traumatic injury. Infact, elevated levels in cTnI in subjects with severe traumatic injuryare often associated with poor outcomes (See, Cai et al., PrognosticValue of Cardiac Troponin I Following Severe Traumatic Brain Injury;Academic Surgical Congress Abstracts 2015, herein incorporated byreference). Given this, the discovery in the present disclosure thatdetecting and/or measuring cTnI levels in a subject who has sustained ormay have sustained an injury to the head can be used to predict theoutcome and severity of injury of a human subject with a mild TBI issurprising.

Specifically, such a method can comprise the steps of determining thelevel of cardiac troponin I in a sample taken from the subject within 28hours, such as within 24 hours, after an injury to the head, andpredicting the subject as having an unfavorable outcome, for example at1 month or 6 months, or having a more severe traumatic brain injury ifthe levels of cTnI are higher than a reference level of the cTnI orpredicting the subject as having a favorable outcome, for example at 1month or 6 months, or having a less severe traumatic brain injury if thelevels of cTnI are lower than a reference level of the cTnI. The samplecan be a biological sample.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, within about 24hours, within about 25 hours, within about 26 hours, within about 27hours, or within about 28 hours of an injury to the head.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cardiac troponin is determined at oneor more time points. In certain embodiments, the subject may besuspected of having a mild traumatic brain injury based on the GlasgowComa Scale score. In certain embodiments, the subject may be suspectedof having a mild traumatic brain injury based on an abnormal head CT. Insome embodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the subject has received a GOSE score after theassay is performed. In some embodiments, the subject is suspected ashaving an unfavorable outcome based on the GOSE score. In someembodiments, the subject has a GOSE score of less than 5 at 1 month, 2months, 3 months, 4 months, 5 months, or 6 months after the suspectedinjury. In some embodiments, the reference level of cTnI is correlatedwith subjects having an unfavorable outcome. In some embodiments, thereference level of cTnI is correlated with a GOSE score of 1-5. In someembodiments, the subject is suspected as having a favorable outcomebased on the GOSE score. In some embodiments, the reference level ofcTnI is correlated with subjects having a favorable outcome. In someembodiments, the reference level of cTnI is correlated with a GOSE scoreof 6-8.

In some embodiments, the reference level of cTnI is correlated withsubjects having a more severe traumatic brain injury, such as moderateto severe traumatic brain injury. In some embodiments, the referencelevel of cTnI is correlated with a Glasgow Coma Scale score of 3-12. Insome embodiments, the subject is suspected as having mild traumaticbrain injury based on the Glasgow Coma Scale score. In some embodiments,the reference level of cTnI is correlated with subjects having a lesssevere traumatic brain injury, such as mild traumatic brain injury. Insome embodiments, the reference level of cTnI is correlated with aGlasgow Coma Scale score of 13-15.

Generally, a reference level of cTnI can also be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In certain embodiments, the reference level may be correlated withcontrol subjects that have not sustained a head injury.

In some embodiments, the method can include obtaining samples from thesubject and contacting the samples with an antibody for cardiac troponinI to allow formation of a complex of the antibody and cardiac troponinI. The method also includes detecting the resulting antibody-cardiactroponin I complex.

In some embodiments, the reference level of cTnI is determined by anassay having a sensitivity of between at least about 65% to about 100%and a specificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 65% to about100%, between at least about 65% to at least about 99%, between at leastabout 65% to at least about 95%, between at least about 65% to at leastabout 90%, between at least about 65% to at least about 85%, between atleast about 65% to at least about 80%, between at least about 65% to atleast about 75%, between at least about 65% to at least about 70%,between at least about 75% to about 100%, between at least about 75% toat least about 99%, between at least about 75% to at least about 95%,between at least about 75% to at least about 90%, between at least about75% to at least about 85%, between at least about 75% to at least about80%, between at least about 80% to about 100%, between at least about80% to at least about 99%, between at least about 80% to at least about95%, between at least about 80% to at least about 90%, between at leastabout 85% to about 100%, between at least about 85% to at least about99%, between at least about 85% to at least about 95%, between at leastabout 85% to at least about 90%, between at least about 95% to about100%, or between at least about 95% to at least about 99%. In someembodiments, the sensitivity is at least about 65.0%, at least about70.0%, at least about 75.0%, at least about 80.0%, at least about 83.0%,at least about 83.3%, at least about 85.0%, at least about 87.5%, atleast about 90.0%, at least about 95.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 45% to about 100%, between at leastabout 45% to about 99%, between at least about 45% to about 95%, betweenat least about 45% to about 90%, between at least about 45% to about85%, between at least about 45% to about 80%, between at least about 45%to about 75%, between at least about 45% to about 70%, between at leastabout 45% to about 60%, between at least about 45% to about 50%, betweenat least about 50% to about 100%, between at least about 50% to about99%, between at least about 50% to about 95%, between at least about 50%to about 90%, between at least about 50% to about 85%, between at leastabout 50% to about 80%, between at least about 50% to about 75%, betweenat least about 50% to about 70%, between at least about 50% to about60%, between at least about 60% to about 100%, between at least about60% to about 99%, between at least about 60% to about 95%, between atleast about 60% to about 90%, between at least about 60% to about 85%,between at least about 60% to about 80%, between at least about 60% toabout 75%, between at least about 60% to about 70%, between at leastabout 70% to about 100%, between at least about 70% to about 99%,between at least about 70% to about 95%, between at least about 70% toabout 90%, between at least about 70% to about 85%, between at leastabout 70% to about 80%, between at least about 70% to about 75%, betweenat least about 80% to about 100%, between at least about 80% to about99%, between at least about 80% to about 95%, between at least about 80%to about 90%, between at least about 80% to about 85%, between at leastabout 90% to about 100%, between at least about 90% to about 99%,between at least about 90% to about 95%, between at least about 95% toabout 99%, or between at least about 95% to about 100. In someembodiments, the specificity is at least about 30.0%, at least about31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%,at least about 35.0%, at least about 36.0%, at least about 37.0%, atleast about 38.0%, at least about 39.0%, at least about 40.0%, at leastabout 45.0%, at least about 49.2%, at least about 50.0%, at least about54.9%, at least about 55.0%, at least about 60.0%, at least about 65.0%,at least about 70.0%, at least about 75.0%, at least about 80.0%, atleast about 85.0%, at least about 90.0%, at least about 91.0%, at leastabout 92.0%, at least about 93.0%, at least about 94.0%, at least about95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%,at least about 99.0%, at least about 99.1%, at least about 99.2%, atleast about 99.3%, at least about 99.4%, at least about 99.5%, at leastabout 99.6%, at least about 99.7%, at least about 99.8%, at least about99.9%, or at least about 100.0%. For example, the sensitivity is atleast about 99% and the specificity is at least about 75%, thesensitivity is at least about 99% and the specificity is at least about99%, or the sensitivity is at least about 100% and the specificity is atleast about 100%.

In some embodiments, the reference level of cardiac troponin I in thesample is from about 1.0 pg/mL to about 50.0 pg/mL, about 1.5 pg/mL toabout 50.0 pg/mL, about 2.0 pg/mL to about 50.0 pg/mL, about 2.5 pg/mLto about 50.0 pg/mL, about 3.0 pg/mL to about 50.0 pg/mL, about 3.5pg/mL to about 50.0 pg/mL, about 4.0 pg/mL to about 50.0 pg/mL, about4.5 pg/mL to about 50.0 pg/mL, about 5.0 pg/mL to about 50.0 pg/mL,about 5.5 pg/mL to about 50.0 pg/mL, about 6.0 pg/mL to about 50.0pg/mL, about 6.5 pg/mL to about 50.0 pg/mL, about 7.0 pg/mL to about50.0 pg/mL, about 7.5 pg/mL to about 50.0 pg/mL, about 8.0 pg/mL toabout 50.0 pg/mL, about 8.5 pg/mL to about 50.0 pg/mL, about 9.0 pg/mLto about 50.0 pg/mL, about 9.5 pg/mL to about 50.0 pg/mL, about 10.0pg/mL to about 50.0 pg/mL, about 1.0 pg/mL to about 40.0 pg/mL, about1.5 pg/mL to about 40.0 pg/mL, about 2.0 pg/mL to about 40.0 pg/mL,about 2.5 pg/mL to about 40.0 pg/mL, about 3.0 pg/mL to about 40.0pg/mL, about 3.5 pg/mL to about 40.0 pg/mL, about 4.0 pg/mL to about40.0 pg/mL, about 4.5 pg/mL to about 40.0 pg/mL, about 5.0 pg/mL toabout 40.0 pg/mL, about 5.5 pg/mL to about 40.0 pg/mL, about 6.0 pg/mLto about 40.0 pg/mL, about 6.5 pg/mL to about 40.0 pg/mL, about 7.0pg/mL to about 40.0 pg/mL, about 7.5 pg/mL to about 40.0 pg/mL, about8.0 pg/mL to about 40.0 pg/mL, about 8.5 pg/mL to about 40.0 pg/mL,about 9.0 pg/mL to about 40.0 pg/mL, about 9.5 pg/mL to about 40.0pg/mL, about 10.0 pg/mL to about 40.0 pg/mL, about 1.0 pg/mL to about35.0 pg/mL, about 1.5 pg/mL to about 35.0 pg/mL, about 2.0 pg/mL toabout 35.0 pg/mL, about 2.5 pg/mL to about 35.0 pg/mL, about 3.0 pg/mLto about 35.0 pg/mL, about 3.5 pg/mL to about 35.0 pg/mL, about 4.0pg/mL to about 35.0 pg/mL, about 4.5 pg/mL to about 35.0 pg/mL, about5.0 pg/mL to about 35.0 pg/mL, about 5.5 pg/mL to about 35.0 pg/mL,about 6.0 pg/mL to about 35.0 pg/mL, about 6.5 pg/mL to about 35.0pg/mL, about 7.0 pg/mL to about 35.0 pg/mL, about 7.5 pg/mL to about35.0 pg/mL, about 8.0 pg/mL to about 35.0 pg/mL, about 8.5 pg/mL toabout 35.0 pg/mL, about 9.0 pg/mL to about 35.0 pg/mL, about 9.5 pg/mLto about 35.0 pg/mL, about 10.0 pg/mL to about 35.0 pg/mL, about 1.0pg/mL to about 30.0 pg/mL, about 1.5 pg/mL to about 30.0 pg/mL, about2.0 pg/mL to about 30.0 pg/mL, about 2.5 pg/mL to about 30.0 pg/mL,about 3.0 pg/mL to about 30.0 pg/mL, about 3.5 pg/mL to about 30.0pg/mL, about 4.0 pg/mL to about 30.0 pg/mL, about 4.5 pg/mL to about30.0 pg/mL, about 5.0 pg/mL to about 30.0 pg/mL, about 5.5 pg/mL toabout 30.0 pg/mL, about 6.0 pg/mL to about 30.0 pg/mL, about 6.5 pg/mLto about 30.0 pg/mL, about 7.0 pg/mL to about 30.0 pg/mL, about 7.5pg/mL to about 30.0 pg/mL, about 8.0 pg/mL to about 30.0 pg/mL, about8.5 pg/mL to about 30.0 pg/mL, about 9.0 pg/mL to about 30.0 pg/mL,about 9.5 pg/mL to about 30.0 pg/mL, about 10.0 pg/mL to about 30.0pg/mL, about 1.0 pg/mL to about 25.0 pg/mL, about 1.5 pg/mL to about25.0 pg/mL, about 2.0 pg/mL to about 25.0 pg/mL, about 2.5 pg/mL toabout 25.0 pg/mL, about 3.0 pg/mL to about 25.0 pg/mL, about 3.5 pg/mLto about 25.0 pg/mL, about 4.0 pg/mL to about 25.0 pg/mL, about 4.5pg/mL to about 25.0 pg/mL, about 5.0 pg/mL to about 25.0 pg/mL, about5.5 pg/mL to about 25.0 pg/mL, about 6.0 pg/mL to about 25.0 pg/mL,about 6.5 pg/mL to about 25.0 pg/mL, about 7.0 pg/mL to about 25.0pg/mL, about 7.5 pg/mL to about 25.0 pg/mL, about 8.0 pg/mL to about25.0 pg/mL, about 8.5 pg/mL to about 25.0 pg/mL, about 9.0 pg/mL toabout 25.0 pg/mL, about 9.5 pg/mL to about 25.0 pg/mL, about 10.0 pg/mLto about 25.0 pg/mL, about 1.0 pg/mL to about 24.0 pg/mL, about 1.5pg/mL to about 24.0 pg/mL, about 2.0 pg/mL to about 24.0 pg/mL, about2.5 pg/mL to about 24.0 pg/mL, about 3.0 pg/mL to about 24.0 pg/mL,about 3.5 pg/mL to about 24.0 pg/mL, about 4.0 pg/mL to about 24.0pg/mL, about 4.5 pg/mL to about 24.0 pg/mL, about 5.0 pg/mL to about24.0 pg/mL, about 5.5 pg/mL to about 24.0 pg/mL, about 6.0 pg/mL toabout 24.0 pg/mL, about 6.5 pg/mL to about 24.0 pg/mL, about 7.0 pg/mLto about 24.0 pg/mL, about 7.5 pg/mL to about 24.0 pg/mL, about 8.0pg/mL to about 24.0 pg/mL, about 8.5 pg/mL to about 24.0 pg/mL, about9.0 pg/mL to about 24.0 pg/mL, about 9.5 pg/mL to about 24.0 pg/mL,about 10.0 pg/mL to about 24.0 pg/mL, about 1.0 pg/mL to about 23.0pg/mL, about 1.5 pg/mL to about 23.0 pg/mL, about 2.0 pg/mL to about23.0 pg/mL, about 2.5 pg/mL to about 23.0 pg/mL, about 3.0 pg/mL toabout 23.0 pg/mL, about 3.5 pg/mL to about 23.0 pg/mL, about 4.0 pg/mLto about 23.0 pg/mL, about 4.5 pg/mL to about 23.0 pg/mL, about 5.0pg/mL to about 23.0 pg/mL, about 5.5 pg/mL to about 23.0 pg/mL, about6.0 pg/mL to about 23.0 pg/mL, about 6.5 pg/mL to about 23.0 pg/mL,about 7.0 pg/mL to about 23.0 pg/mL, about 7.5 pg/mL to about 23.0pg/mL, about 8.0 pg/mL to about 23.0 pg/mL, about 8.5 pg/mL to about23.0 pg/mL, about 9.0 pg/mL to about 23.0 pg/mL, about 9.5 pg/mL toabout 23.0 pg/mL, about 10.0 pg/mL to about 23.0 pg/mL, about 1.0 pg/mLto about 22.0 pg/mL, about 1.5 pg/mL to about 22.0 pg/mL, about 2.0pg/mL to about 22.0 pg/mL, about 2.5 pg/mL to about 22.0 pg/mL, about3.0 pg/mL to about 22.0 pg/mL, about 3.5 pg/mL to about 22.0 pg/mL,about 4.0 pg/mL to about 22.0 pg/mL, about 4.5 pg/mL to about 22.0pg/mL, about 5.0 pg/mL to about 22.0 pg/mL, about 5.5 pg/mL to about22.0 pg/mL, about 6.0 pg/mL to about 22.0 pg/mL, about 6.5 pg/mL toabout 22.0 pg/mL, about 7.0 pg/mL to about 22.0 pg/mL, about 7.5 pg/mLto about 22.0 pg/mL, about 8.0 pg/mL to about 22.0 pg/mL, about 8.5pg/mL to about 22.0 pg/mL, about 9.0 pg/mL to about 22.0 pg/mL, about9.5 pg/mL to about 22.0 pg/mL, about 10.0 pg/mL to about 22.0 pg/mL,about 1.0 pg/mL to about 21.0 pg/mL, about 1.5 pg/mL to about 21.0pg/mL, about 2.0 pg/mL to about 21.0 pg/mL, about 2.5 pg/mL to about21.0 pg/mL, about 3.0 pg/mL to about 21.0 pg/mL, about 3.5 pg/mL toabout 21.0 pg/mL, about 4.0 pg/mL to about 21.0 pg/mL, about 4.5 pg/mLto about 21.0 pg/mL, about 5.0 pg/mL to about 21.0 pg/mL, about 5.5pg/mL to about 21.0 pg/mL, about 6.0 pg/mL to about 21.0 pg/mL, about6.5 pg/mL to about 21.0 pg/mL, about 7.0 pg/mL to about 21.0 pg/mL,about 7.5 pg/mL to about 21.0 pg/mL, about 8.0 pg/mL to about 21.0pg/mL, about 8.5 pg/mL to about 21.0 pg/mL, about 9.0 pg/mL to about21.0 pg/mL, about 9.5 pg/mL to about 21.0 pg/mL, about 10.0 pg/mL toabout 21.0 pg/mL, about 1.0 pg/mL to about 20.0 pg/mL, about 1.5 pg/mLto about 20.0 pg/mL, about 2.0 pg/mL to about 20.0 pg/mL, about 2.5pg/mL to about 20.0 pg/mL, about 3.0 pg/mL to about 20.0 pg/mL, about3.5 pg/mL to about 20.0 pg/mL, about 4.0 pg/mL to about 20.0 pg/mL,about 4.5 pg/mL to about 20.0 pg/mL, about 5.0 pg/mL to about 20.0pg/mL, about 5.5 pg/mL to about 20.0 pg/mL, about 6.0 pg/mL to about20.0 pg/mL, about 6.5 pg/mL to about 20.0 pg/mL, about 7.0 pg/mL toabout 20.0 pg/mL, about 7.5 pg/mL to about 20.0 pg/mL, about 8.0 pg/mLto about 20.0 pg/mL, about 8.5 pg/mL to about 20.0 pg/mL, about 9.0pg/mL to about 20.0 pg/mL, about 9.5 pg/mL to about 20.0 pg/mL, about10.0 pg/mL to about 20.0 pg/mL, about 1.0 pg/mL to about 19.0 pg/mL,about 1.5 pg/mL to about 19.0 pg/mL, about 2.0 pg/mL to about 19.0pg/mL, about 2.5 pg/mL to about 19.0 pg/mL, about 3.0 pg/mL to about19.0 pg/mL, about 3.5 pg/mL to about 19.0 pg/mL, about 4.0 pg/mL toabout 19.0 pg/mL, about 4.5 pg/mL to about 19.0 pg/mL, about 5.0 pg/mLto about 19.0 pg/mL, about 5.5 pg/mL to about 19.0 pg/mL, about 6.0pg/mL to about 19.0 pg/mL, about 6.5 pg/mL to about 19.0 pg/mL, about7.0 pg/mL to about 19.0 pg/mL, about 7.5 pg/mL to about 19.0 pg/mL,about 8.0 pg/mL to about 19.0 pg/mL, about 8.5 pg/mL to about 19.0pg/mL, about 9.0 pg/mL to about 19.0 pg/mL, about 9.5 pg/mL to about19.0 pg/mL, about 10.0 pg/mL to about 19.0 pg/mL, about 1.0 pg/mL toabout 18.0 pg/mL, about 1.5 pg/mL to about 18.0 pg/mL, about 2.0 pg/mLto about 18.0 pg/mL, about 2.5 pg/mL to about 18.0 pg/mL, about 3.0pg/mL to about 18.0 pg/mL, about 3.5 pg/mL to about 18.0 pg/mL, about4.0 pg/mL to about 18.0 pg/mL, about 4.5 pg/mL to about 18.0 pg/mL,about 5.0 pg/mL to about 18.0 pg/mL, about 5.5 pg/mL to about 18.0pg/mL, about 6.0 pg/mL to about 18.0 pg/mL, about 6.5 pg/mL to about18.0 pg/mL, about 7.0 pg/mL to about 18.0 pg/mL, about 7.5 pg/mL toabout 18.0 pg/mL, about 8.0 pg/mL to about 18.0 pg/mL, about 8.5 pg/mLto about 18.0 pg/mL, about 9.0 pg/mL to about 18.0 pg/mL, about 9.5pg/mL to about 18.0 pg/mL, about 10.0 pg/mL to about 18.0 pg/mL, about1.0 pg/mL to about 17.0 pg/mL, about 1.5 pg/mL to about 17.0 pg/mL,about 2.0 pg/mL to about 17.0 pg/mL, about 2.5 pg/mL to about 17.0pg/mL, about 3.0 pg/mL to about 17.0 pg/mL, about 3.5 pg/mL to about17.0 pg/mL, about 4.0 pg/mL to about 17.0 pg/mL, about 4.5 pg/mL toabout 17.0 pg/mL, about 5.0 pg/mL to about 17.0 pg/mL, about 5.5 pg/mLto about 17.0 pg/mL, about 6.0 pg/mL to about 17.0 pg/mL, about 6.5pg/mL to about 17.0 pg/mL, about 7.0 pg/mL to about 17.0 pg/mL, about7.5 pg/mL to about 17.0 pg/mL, about 8.0 pg/mL to about 17.0 pg/mL,about 8.5 pg/mL to about 17.0 pg/mL, about 9.0 pg/mL to about 17.0pg/mL, about 9.5 pg/mL to about 17.0 pg/mL, about 10.0 pg/mL to about17.0 pg/mL, about 1.0 pg/mL to about 16.0 pg/mL, about 1.5 pg/mL toabout 16.0 pg/mL, about 2.0 pg/mL to about 16.0 pg/mL, about 2.5 pg/mLto about 16.0 pg/mL, about 3.0 pg/mL to about 16.0 pg/mL, about 3.5pg/mL to about 16.0 pg/mL, about 4.0 pg/mL to about 16.0 pg/mL, about4.5 pg/mL to about 16.0 pg/mL, about 5.0 pg/mL to about 16.0 pg/mL,about 5.5 pg/mL to about 16.0 pg/mL, about 6.0 pg/mL to about 16.0pg/mL, about 6.5 pg/mL to about 16.0 pg/mL, about 7.0 pg/mL to about16.0 pg/mL, about 7.5 pg/mL to about 16.0 pg/mL, about 8.0 pg/mL toabout 16.0 pg/mL, about 8.5 pg/mL to about 16.0 pg/mL, about 9.0 pg/mLto about 16.0 pg/mL, about 9.5 pg/mL to about 16.0 pg/mL, about 10.0pg/mL to about 16.0 pg/mL, about 1.0 pg/mL to about 15.0 pg/mL, about1.5 pg/mL to about 15.0 pg/mL, about 2.0 pg/mL to about 15.0 pg/mL,about 2.5 pg/mL to about 15.0 pg/mL, about 3.0 pg/mL to about 15.0pg/mL, about 3.5 pg/mL to about 15.0 pg/mL, about 4.0 pg/mL to about15.0 pg/mL, about 4.5 pg/mL to about 15.0 pg/mL, about 5.0 pg/mL toabout 15.0 pg/mL, about 5.5 pg/mL to about 15.0 pg/mL, about 6.0 pg/mLto about 15.0 pg/mL, about 6.5 pg/mL to about 15.0 pg/mL, about 7.0pg/mL to about 15.0 pg/mL, about 7.5 pg/mL to about 15.0 pg/mL, about8.0 pg/mL to about 15.0 pg/mL, about 8.5 pg/mL to about 15.0 pg/mL,about 9.0 pg/mL to about 15.0 pg/mL, about 9.5 pg/mL to about 15.0 pg/mLor about 10.0 pg/mL to about 15.0 pg/mL. In some embodiments, the amountof cardiac troponin I in the sample is about 1.0 pg/mL, about 1.5 pg/mL,about 2.0 pg/mL, about 2.5 pg/mL, about 3.0 pg/mL, about 3.5 pg/mL,about 4.0 pg/mL, about 4.5 pg/mL, about 5.0 pg/mL, about 5.5 pg/mL,about 5.6 pg/mL, about 5.7 pg/mL, about 5.8 pg/mL, about 5.9 pg/mL,about 6.0 pg/mL, about 6.5 pg/mL, about 7.0 pg/mL, about 7.5 pg/mL,about 8.0 pg/mL, about 8.5 pg/mL, about 9.0 pg/mL, about 9.5 pg/mL, orabout 10.0 pg/mL.

In some embodiments, the method further includes treating the humansubject predicted as having an unfavorable outcome with a traumaticbrain injury treatment, as described below. In some embodiments, themethod further includes monitoring the human subject predicted as havingan unfavorable outcome, as described below. In some embodiments, themethod further includes ordering additional tests to obtain furtherclinical information about the mild TBI in the human subject predictedas having an unfavorable outcome. In some embodiments, the methodincludes treating the human subject predicted to have an unfavorableoutcome with a cardioprotective treatment to protect the heart asdescribed below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assaysare described in further detail herein in Sections 11-13. Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. For example, a clinical chemistryformat can include an assay that involves one antibody or no antibody.Examples of analyzers that can be used for the clinical chemistry formatare described in U.S. Patent publication Nos. 2016/0320422 and2015/0112630.

7. Methods of Aiding in Predicting or Predicting the Outcome of a HumanSubject Having Mild Traumatic Brain Injury based on Changes in CardiacTroponin I (cTnI) Levels

The present disclosure relates, among other methods, to a method ofaiding in predicting (or predicting) the outcome of a human subjecthaving mild traumatic brain injury (TBI), e.g., determining whether thesubject will have an unfavorable or negative outcome or favorableoutcome. As used herein, the phrase “determining whether the subject hasa favorable outcome” refers to the fact that the aforementioned methodcan be used, e.g., with other information (e.g., clinical assessmentdata), to determine that the subject is more likely than not to have apositive outcome from the mild TBI. Additionally, as used herein, thephrase “determining whether the subject has an unfavorable outcome”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have an unfavorable ornegative outcome from the mild TBI

As mentioned above, the methods described herein can be used todetermine whether a subject diagnosed with a mild TBI is more likelythan not to have (1) a favorable outcome (optionally, the favorableoutcome can be that the subject fully recovers and does not continue toexperience one or more symptoms of a mild TBI); or (2) an unfavorableoutcome (optionally, the unfavorable outcome can be that the subjectdoes not fully recover and does continue to experience one or moresymptoms of a mild TBI).

Alternatively and optionally, a favorable outcome can mean that thesubject is more likely than not to suffer no more than onepost-concussion syndrome symptom as a result of the mild TBI such as:(a) physical difficulties (e.g., headaches, dizziness, fatigue,sensitivity to light noise and light, etc.); (b) cognitive difficulties(e.g., trouble concentration, memory problems, restlessness, etc.); (c)emotional difficulties (e.g., personality changes, irritability,depression, apathy, etc.); or (d) sleep difficulties (e.g., insomnia,etc.). Alternatively and option, subject who have an unfavorable outcomeare more likely to suffer from more than one post-concussion syndromesymptom such as: (a) physical difficulties (e.g., headaches, dizziness,fatigue, sensitivity to light noise and light, etc.); (b) cognitivedifficulties (e.g., trouble concentration, memory problems,restlessness, etc.); (c) emotional difficulties (e.g, personalitychanges, irritability, depression, apathy, etc.); (d) sleep difficulties(e.g., insomnia, etc.); or (e) any combinations of (a)-(d)).Alternatively and optionally, an unfavorable outcome can also mean thata subject exhibits one or more symptoms of mild TBI. Alternatively andoptionally, an unfavorable outcome can also mean that the subject'sconditions worsens from mild TBI to moderate, moderate to severe orsevere. Additionally, subjects having a favorable outcome are likely tohave a GOSE score of 5 or greater whereas subjects having an unfavorableoutcome are likely to have a GOSE score of less than 5.

The methods disclosed herein can include performing at least two or moreassays on one or more samples obtained from the human subject withinafter an injury to the head at two or more different time points anddetermining the level of cardiac troponin I at each of these timepoints. If the amount or level of cardiac troponin I in the sample hasincreased from the first (or previous) sample to the subsequent (e.g.,second) sample, then a determination is made that the subject ispredicted to have an unfavorable outcome, for example at 6 months, or amore severe traumatic brain injury. However, if the amount or level ofcardiac troponin I in the sample has remained the same or decreased fromthe first (or previous) sample to the second (or subsequent) sample,then a determination is made that the subject is predicted to have afavorable outcome at 6 months or a less severe traumatic brain injury.

Specifically, such a method can comprise the steps of: (a) performing anassay on samples from the human subject to measure or detect a level ofcardiac troponin I in a first sample and a second sample, wherein thefirst sample is taken from the human subject at a first time pointwithin 24 hours after an injury to the head and the second sample istaken from the human subject about 0 hours to 6 hours after the firstsample, wherein the samples are biological samples; (b) determiningwhether the amount of cardiac troponin I has increased or decreased fromthe first sample to the second sample; and (c) predicting an unfavorableor negative outcome in the human subject if the level of cardiactroponin I detected has increased from the first sample to the secondsample. In some embodiments, a human subject is predicted as having anunfavorable or negative outcome if there is an increase by at least anabsolute amount from the first sample to the second sample. In someembodiments, a human subject is predicted as having an unfavorable ornegative outcome if the level of cardiac troponin I detected hasincreased by at least about 20% from the first sample to the secondsample, and a human subject is predicted as having a favorable outcomein the human subject if the level of cardiac troponin I detected hasremained unchanged, decreased, or increased, such as increased less thanabout 20% from the first sample to the second sample.

The first (or previous) sample may be obtained or taken from the subject0 minutes, within about 1 minute, within about 2 minutes, within about 3minutes, within about 4 minutes, within about 5 minutes, within about 6minutes, within about 7 minutes, within about 8 minutes, within about 9minutes, within about 10 minutes, within about 11 minutes, within about12 minutes, within about 13 minutes, within about 14 minutes, withinabout 15 minutes, within about 20 minutes, within about 30 minutes,within about 1 hour, within about 2 hours, within about 3 hours, withinabout 4 hours, within about 5 hours, within about 6 hours, within about7 hours, within about 8 hours, within about 9 hours, within about 10hours, within about 11 hours, within about 12 hours, within about 13hours, within about 14 hours, within about 15 hours, within about 16hours, within about 17 hours, within about 18 hours, within about 19hours, within about 20 hours, within about 21 hours, within about 22hours, within about 23 hours or within about 24 hours of an injury tothe head. In some embodiments, the (or previous) sample is taken withinabout 0 to about 1 hours, within about 0 to about 2 hours, within about0 to about 3 hours, within about 0 to about 4 hours, within about 0 toabout 5 hours, within about 0 to about 6 hours, within about 0 to about7 hours, within about 0 to about 8 hours, within about 0 to about 9hours, within about 0 to about 10 hours, within about 0 to about 11hours, within about 0 to about 12 hours, within about 0 to about 18hours, within about 6 to about 12 hours, within about 12 to about 18hours, within about 18 to about 24 hours, or greater than 24 hours afterthe suspected injury. The second (or subsequent) sample can be obtainedafter about 0 hour to 6 hours after the first (or last) sample wasobtained. In some embodiments, the second (or subsequent) sample istaken from the subject within at least about 30 minutes, within at leastabout 1 hour, within at least about 2 hours, within at least about 3hours, within at least about 4 hours, within at least about 5 hours, orwithin at least about 6 hours after the first sample is taken from thesubject.

In some embodiments, the subject is predicted as having an unfavorableoutcome if the level of cardiac troponin I detected has increased fromthe first sample to the second sample. For example, the subject can bepredicted as having an unfavorable outcome if the level of cardiactroponin I detected has increased from the first sample to the secondsample by at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 100%, at least about 250%, at least about 500%, or at leastabout 1000%. In some embodiments, the subject can be predicted as havingan unfavorable outcome if the level of cardiac troponin I detected hasincreased from the first sample to the second sample by at least about20%.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cardiac troponin is determined at oneor more time points. In certain embodiments, the subject may besuspected of having a mild traumatic brain injury based on the GlasgowComa Scale score. In certain embodiments, the subject may be suspectedof having a mild traumatic brain injury based on an abnormal head CT. Insome embodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the subject has received a GOSE score after theassay is performed. In some embodiments, the subject is suspected ashaving an unfavorable outcome based on the GOSE score. In someembodiments, the subject has a GOSE score of less than 5 at 1 month, 2months, 3 months, 4 months, 5 months, or 6 months after the suspectedinjury. In some embodiments, an increased level of cTnI is correlatedwith subjects having an unfavorable outcome. In some embodiments, theincreased level of cTnI is correlated with a GOSE score of 1-5. In someembodiments, the subject is suspected as having a favorable outcomebased on the GOSE score. In some embodiments, a decreased or unchangedlevel of cTnI is correlated with subjects having a favorable outcome. Insome embodiments, the decreased or unchanged level of cTnI is correlatedwith a GOSE score of 6-8.

In some embodiments, an increased level of cTnI is correlated withsubjects having a more severe traumatic brain injury, such as moderateto severe traumatic brain injury. In some embodiments, the increasedlevel of cTnI is correlated with a Glasgow Coma Scale score of 3-12. Insome embodiments, the subject is suspected as having mild traumaticbrain injury based on the Glasgow Coma Scale score. In some embodiments,a decreased or unchanged level of cTnI is correlated with subjectshaving a less severe traumatic brain injury, such as mild traumaticbrain injury. In some embodiments, the decreased or unchanged level ofcTnI is correlated with a Glasgow Coma Scale score of 13-15.

In some embodiments, the method can include obtaining samples from thesubject and contacting the samples with an antibody for cardiac troponinI to allow formation of a complex of the antibody and cardiac troponinI. The method also includes detecting the resulting antibody-cardiactroponin I complex.

In some embodiments, the amount of cardiac troponin I in the first orprevious sample is from about 1.0 pg/mL to about 50.0 pg/mL, about 1.5pg/mL to about 50.0 pg/mL, about 2.0 pg/mL to about 50.0 pg/mL, about2.5 pg/mL to about 50.0 pg/mL, about 3.0 pg/mL to about 50.0 pg/mL,about 3.5 pg/mL to about 50.0 pg/mL, about 4.0 pg/mL to about 50.0pg/mL, about 4.5 pg/mL to about 50.0 pg/mL, about 5.0 pg/mL to about50.0 pg/mL, about 5.5 pg/mL to about 50.0 pg/mL, about 6.0 pg/mL toabout 50.0 pg/mL, about 6.5 pg/mL to about 50.0 pg/mL, about 7.0 pg/mLto about 50.0 pg/mL, about 7.5 pg/mL to about 50.0 pg/mL, about 8.0pg/mL to about 50.0 pg/mL, about 8.5 pg/mL to about 50.0 pg/mL, about9.0 pg/mL to about 50.0 pg/mL, about 9.5 pg/mL to about 50.0 pg/mL,about 10.0 pg/mL to about 50.0 pg/mL, about 1.0 pg/mL to about 40.0pg/mL, about 1.5 pg/mL to about 40.0 pg/mL, about 2.0 pg/mL to about40.0 pg/mL, about 2.5 pg/mL to about 40.0 pg/mL, about 3.0 pg/mL toabout 40.0 pg/mL, about 3.5 pg/mL to about 40.0 pg/mL, about 4.0 pg/mLto about 40.0 pg/mL, about 4.5 pg/mL to about 40.0 pg/mL, about 5.0pg/mL to about 40.0 pg/mL, about 5.5 pg/mL to about 40.0 pg/mL, about6.0 pg/mL to about 40.0 pg/mL, about 6.5 pg/mL to about 40.0 pg/mL,about 7.0 pg/mL to about 40.0 pg/mL, about 7.5 pg/mL to about 40.0pg/mL, about 8.0 pg/mL to about 40.0 pg/mL, about 8.5 pg/mL to about40.0 pg/mL, about 9.0 pg/mL to about 40.0 pg/mL, about 9.5 pg/mL toabout 40.0 pg/mL, about 10.0 pg/mL to about 40.0 pg/mL, about 1.0 pg/mLto about 35.0 pg/mL, about 1.5 pg/mL to about 35.0 pg/mL, about 2.0pg/mL to about 35.0 pg/mL, about 2.5 pg/mL to about 35.0 pg/mL, about3.0 pg/mL to about 35.0 pg/mL, about 3.5 pg/mL to about 35.0 pg/mL,about 4.0 pg/mL to about 35.0 pg/mL, about 4.5 pg/mL to about 35.0pg/mL, about 5.0 pg/mL to about 35.0 pg/mL, about 5.5 pg/mL to about35.0 pg/mL, about 6.0 pg/mL to about 35.0 pg/mL, about 6.5 pg/mL toabout 35.0 pg/mL, about 7.0 pg/mL to about 35.0 pg/mL, about 7.5 pg/mLto about 35.0 pg/mL, about 8.0 pg/mL to about 35.0 pg/mL, about 8.5pg/mL to about 35.0 pg/mL, about 9.0 pg/mL to about 35.0 pg/mL, about9.5 pg/mL to about 35.0 pg/mL, about 10.0 pg/mL to about 35.0 pg/mL,about 1.0 pg/mL to about 30.0 pg/mL, about 1.5 pg/mL to about 30.0pg/mL, about 2.0 pg/mL to about 30.0 pg/mL, about 2.5 pg/mL to about30.0 pg/mL, about 3.0 pg/mL to about 30.0 pg/mL, about 3.5 pg/mL toabout 30.0 pg/mL, about 4.0 pg/mL to about 30.0 pg/mL, about 4.5 pg/mLto about 30.0 pg/mL, about 5.0 pg/mL to about 30.0 pg/mL, about 5.5pg/mL to about 30.0 pg/mL, about 6.0 pg/mL to about 30.0 pg/mL, about6.5 pg/mL to about 30.0 pg/mL, about 7.0 pg/mL to about 30.0 pg/mL,about 7.5 pg/mL to about 30.0 pg/mL, about 8.0 pg/mL to about 30.0pg/mL, about 8.5 pg/mL to about 30.0 pg/mL, about 9.0 pg/mL to about30.0 pg/mL, about 9.5 pg/mL to about 30.0 pg/mL, about 10.0 pg/mL toabout 30.0 pg/mL, about 1.0 pg/mL to about 25.0 pg/mL, about 1.5 pg/mLto about 25.0 pg/mL, about 2.0 pg/mL to about 25.0 pg/mL, about 2.5pg/mL to about 25.0 pg/mL, about 3.0 pg/mL to about 25.0 pg/mL, about3.5 pg/mL to about 25.0 pg/mL, about 4.0 pg/mL to about 25.0 pg/mL,about 4.5 pg/mL to about 25.0 pg/mL, about 5.0 pg/mL to about 25.0pg/mL, about 5.5 pg/mL to about 25.0 pg/mL, about 6.0 pg/mL to about25.0 pg/mL, about 6.5 pg/mL to about 25.0 pg/mL, about 7.0 pg/mL toabout 25.0 pg/mL, about 7.5 pg/mL to about 25.0 pg/mL, about 8.0 pg/mLto about 25.0 pg/mL, about 8.5 pg/mL to about 25.0 pg/mL, about 9.0pg/mL to about 25.0 pg/mL, about 9.5 pg/mL to about 25.0 pg/mL, about10.0 pg/mL to about 25.0 pg/mL, about 1.0 pg/mL to about 24.0 pg/mL,about 1.5 pg/mL to about 24.0 pg/mL, about 2.0 pg/mL to about 24.0pg/mL, about 2.5 pg/mL to about 24.0 pg/mL, about 3.0 pg/mL to about24.0 pg/mL, about 3.5 pg/mL to about 24.0 pg/mL, 4.0 pg/mL to about 24.0pg/mL, about 4.5 pg/mL to about 24.0 pg/mL, about 5.0 pg/mL to about24.0 pg/mL, about 5.5 pg/mL to about 24.0 pg/mL, about 6.0 pg/mL toabout 24.0 pg/mL, about 6.5 pg/mL to about 24.0 pg/mL, about 7.0 pg/mLto about 24.0 pg/mL, about 7.5 pg/mL to about 24.0 pg/mL, about 8.0pg/mL to about 24.0 pg/mL, about 8.5 pg/mL to about 24.0 pg/mL, about9.0 pg/mL to about 24.0 pg/mL, about 9.5 pg/mL to about 24.0 pg/mL,about 10.0 pg/mL to about 24.0 pg/mL, about 1.0 pg/mL to about 23.0pg/mL, about 1.5 pg/mL to about 23.0 pg/mL, about 2.0 pg/mL to about23.0 pg/mL, about 2.5 pg/mL to about 23.0 pg/mL, about 3.0 pg/mL toabout 23.0 pg/mL, about 3.5 pg/mL to about 23.0 pg/mL, about 4.0 pg/mLto about 23.0 pg/mL, about 4.5 pg/mL to about 23.0 pg/mL, about 5.0pg/mL to about 23.0 pg/mL, about 5.5 pg/mL to about 23.0 pg/mL, about6.0 pg/mL to about 23.0 pg/mL, about 6.5 pg/mL to about 23.0 pg/mL,about 7.0 pg/mL to about 23.0 pg/mL, about 7.5 pg/mL to about 23.0pg/mL, about 8.0 pg/mL to about 23.0 pg/mL, about 8.5 pg/mL to about23.0 pg/mL, about 9.0 pg/mL to about 23.0 pg/mL, about 9.5 pg/mL toabout 23.0 pg/mL, about 10.0 pg/mL to about 23.0 pg/mL, about 1.0 pg/mLto about 22.0 pg/mL, about 1.5 pg/mL to about 22.0 pg/mL, about 2.0pg/mL to about 22.0 pg/mL, about 2.5 pg/mL to about 22.0 pg/mL, about3.0 pg/mL to about 22.0 pg/mL, about 3.5 pg/mL to about 22.0 pg/mL,about 4.0 pg/mL to about 22.0 pg/mL, about 4.5 pg/mL to about 22.0pg/mL, about 5.0 pg/mL to about 22.0 pg/mL, about 5.5 pg/mL to about22.0 pg/mL, about 6.0 pg/mL to about 22.0 pg/mL, about 6.5 pg/mL toabout 22.0 pg/mL, about 7.0 pg/mL to about 22.0 pg/mL, about 7.5 pg/mLto about 22.0 pg/mL, about 8.0 pg/mL to about 22.0 pg/mL, about 8.5pg/mL to about 22.0 pg/mL, about 9.0 pg/mL to about 22.0 pg/mL, about9.5 pg/mL to about 22.0 pg/mL, about 10.0 pg/mL to about 22.0 pg/mL,about 1.0 pg/mL to about 21.0 pg/mL, about 1.5 pg/mL to about 21.0pg/mL, about 2.0 pg/mL to about 21.0 pg/mL, about 2.5 pg/mL to about21.0 pg/mL, about 3.0 pg/mL to about 21.0 pg/mL, about 3.5 pg/mL toabout 21.0 pg/mL, about 4.0 pg/mL to about 21.0 pg/mL, about 4.5 pg/mLto about 21.0 pg/mL, about 5.0 pg/mL to about 21.0 pg/mL, about 5.5pg/mL to about 21.0 pg/mL, about 6.0 pg/mL to about 21.0 pg/mL, about6.5 pg/mL to about 21.0 pg/mL, about 7.0 pg/mL to about 21.0 pg/mL,about 7.5 pg/mL to about 21.0 pg/mL, about 8.0 pg/mL to about 21.0pg/mL, about 8.5 pg/mL to about 21.0 pg/mL, about 9.0 pg/mL to about21.0 pg/mL, about 9.5 pg/mL to about 21.0 pg/mL, about 10.0 pg/mL toabout 21.0 pg/mL, about 1.0 pg/mL to about 20.0 pg/mL, about 1.5 pg/mLto about 20.0 pg/mL, about 2.0 pg/mL to about 20.0 pg/mL, about 2.5pg/mL to about 20.0 pg/mL, about 3.0 pg/mL to about 20.0 pg/mL, about3.5 pg/mL to about 20.0 pg/mL, about 4.0 pg/mL to about 20.0 pg/mL,about 4.5 pg/mL to about 20.0 pg/mL, about 5.0 pg/mL to about 20.0pg/mL, about 5.5 pg/mL to about 20.0 pg/mL, about 6.0 pg/mL to about20.0 pg/mL, about 6.5 pg/mL to about 20.0 pg/mL, about 7.0 pg/mL toabout 20.0 pg/mL, about 7.5 pg/mL to about 20.0 pg/mL, about 8.0 pg/mLto about 20.0 pg/mL, about 8.5 pg/mL to about 20.0 pg/mL, about 9.0pg/mL to about 20.0 pg/mL, about 9.5 pg/mL to about 20.0 pg/mL, about10.0 pg/mL to about 20.0 pg/mL, about 1.0 pg/mL to about 19.0 pg/mL,about 1.5 pg/mL to about 19.0 pg/mL, about 2.0 pg/mL to about 19.0pg/mL, about 2.5 pg/mL to about 19.0 pg/mL, about 3.0 pg/mL to about19.0 pg/mL, about 3.5 pg/mL to about 19.0 pg/mL, about 4.0 pg/mL toabout 19.0 pg/mL, about 4.5 pg/mL to about 19.0 pg/mL, about 5.0 pg/mLto about 19.0 pg/mL, about 5.5 pg/mL to about 19.0 pg/mL, about 6.0pg/mL to about 19.0 pg/mL, about 6.5 pg/mL to about 19.0 pg/mL, about7.0 pg/mL to about 19.0 pg/mL, about 7.5 pg/mL to about 19.0 pg/mL,about 8.0 pg/mL to about 19.0 pg/mL, about 8.5 pg/mL to about 19.0pg/mL, about 9.0 pg/mL to about 19.0 pg/mL, about 9.5 pg/mL to about19.0 pg/mL, about 10.0 pg/mL to about 19.0 pg/mL, about 1.0 pg/mL toabout 18.0 pg/mL, about 1.5 pg/mL to about 18.0 pg/mL, about 2.0 pg/mLto about 18.0 pg/mL, about 2.5 pg/mL to about 18.0 pg/mL, about 3.0pg/mL to about 18.0 pg/mL, about 3.5 pg/mL to about 18.0 pg/mL, about4.0 pg/mL to about 18.0 pg/mL, about 4.5 pg/mL to about 18.0 pg/mL,about 5.0 pg/mL to about 18.0 pg/mL, about 5.5 pg/mL to about 18.0pg/mL, about 6.0 pg/mL to about 18.0 pg/mL, about 6.5 pg/mL to about18.0 pg/mL, about 7.0 pg/mL to about 18.0 pg/mL, about 7.5 pg/mL toabout 18.0 pg/mL, about 8.0 pg/mL to about 18.0 pg/mL, about 8.5 pg/mLto about 18.0 pg/mL, about 9.0 pg/mL to about 18.0 pg/mL, about 9.5pg/mL to about 18.0 pg/mL, about 10.0 pg/mL to about 18.0 pg/mL, about1.0 pg/mL to about 17.0 pg/mL, about 1.5 pg/mL to about 17.0 pg/mL,about 2.0 pg/mL to about 17.0 pg/mL, about 2.5 pg/mL to about 17.0pg/mL, about 3.0 pg/mL to about 17.0 pg/mL, about 3.5 pg/mL to about17.0 pg/mL, about 4.0 pg/mL to about 17.0 pg/mL, about 4.5 pg/mL toabout 17.0 pg/mL, about 5.0 pg/mL to about 17.0 pg/mL, about 5.5 pg/mLto about 17.0 pg/mL, about 6.0 pg/mL to about 17.0 pg/mL, about 6.5pg/mL to about 17.0 pg/mL, about 7.0 pg/mL to about 17.0 pg/mL, about7.5 pg/mL to about 17.0 pg/mL, about 8.0 pg/mL to about 17.0 pg/mL,about 8.5 pg/mL to about 17.0 pg/mL, about 9.0 pg/mL to about 17.0pg/mL, about 9.5 pg/mL to about 17.0 pg/mL, about 10.0 pg/mL to about17.0 pg/mL, about 1.0 pg/mL to about 16.0 pg/mL, about 1.5 pg/mL toabout 16.0 pg/mL, about 2.0 pg/mL to about 16.0 pg/mL, about 2.5 pg/mLto about 16.0 pg/mL, about 3.0 pg/mL to about 16.0 pg/mL, about 3.5pg/mL to about 16.0 pg/mL, about 4.0 pg/mL to about 16.0 pg/mL, about4.5 pg/mL to about 16.0 pg/mL, about 5.0 pg/mL to about 16.0 pg/mL,about 5.5 pg/mL to about 16.0 pg/mL, about 6.0 pg/mL to about 16.0pg/mL, about 6.5 pg/mL to about 16.0 pg/mL, about 7.0 pg/mL to about16.0 pg/mL, about 7.5 pg/mL to about 16.0 pg/mL, about 8.0 pg/mL toabout 16.0 pg/mL, about 8.5 pg/mL to about 16.0 pg/mL, about 9.0 pg/mLto about 16.0 pg/mL, about 9.5 pg/mL to about 16.0 pg/mL, about 10.0pg/mL to about 16.0 pg/mL, about 1.0 pg/mL to about 15.0 pg/mL, about1.5 pg/mL to about 15.0 pg/mL, about 2.0 pg/mL to about 15.0 pg/mL,about 2.5 pg/mL to about 15.0 pg/mL, about 3.0 pg/mL to about 15.0pg/mL, about 3.5 pg/mL to about 15.0 pg/mL, about 4.0 pg/mL to about15.0 pg/mL, about 4.5 pg/mL to about 15.0 pg/mL, about 5.0 pg/mL toabout 15.0 pg/mL, about 5.5 pg/mL to about 15.0 pg/mL, about 6.0 pg/mLto about 15.0 pg/mL, about 6.5 pg/mL to about 15.0 pg/mL, about 7.0pg/mL to about 15.0 pg/mL, about 7.5 pg/mL to about 15.0 pg/mL, about8.0 pg/mL to about 15.0 pg/mL, about 8.5 pg/mL to about 15.0 pg/mL,about 9.0 pg/mL to about 15.0 pg/mL, about 9.5 pg/mL to about 15.0 pg/mLor about 10.0 pg/mL to about 15.0 pg/mL.

In some embodiments, the amount of cardiac troponin I in the second orsubsequent sample is from about 1.0 pg/mL to about 50.0 pg/mL, about 1.5pg/mL to about 50.0 pg/mL, about 2.0 pg/mL to about 50.0 pg/mL, about2.5 pg/mL to about 50.0 pg/mL, about 3.0 pg/mL to about 50.0 pg/mL,about 3.5 pg/mL to about 50.0 pg/mL, about 4.0 pg/mL to about 50.0pg/mL, about 4.5 pg/mL to about 50.0 pg/mL, about 5.0 pg/mL to about50.0 pg/mL, about 5.5 pg/mL to about 50.0 pg/mL, about 6.0 pg/mL toabout 50.0 pg/mL, about 6.5 pg/mL to about 50.0 pg/mL, about 7.0 pg/mLto about 50.0 pg/mL, about 7.5 pg/mL to about 50.0 pg/mL, about 8.0pg/mL to about 50.0 pg/mL, about 8.5 pg/mL to about 50.0 pg/mL, about9.0 pg/mL to about 50.0 pg/mL, about 9.5 pg/mL to about 50.0 pg/mL,about 10.0 pg/mL to about 50.0 pg/mL, about 1.0 pg/mL to about 40.0pg/mL, about 1.5 pg/mL to about 40.0 pg/mL, about 2.0 pg/mL to about40.0 pg/mL, about 2.5 pg/mL to about 40.0 pg/mL, about 3.0 pg/mL toabout 40.0 pg/mL, about 3.5 pg/mL to about 40.0 pg/mL, about 4.0 pg/mLto about 40.0 pg/mL, about 4.5 pg/mL to about 40.0 pg/mL, about 5.0pg/mL to about 40.0 pg/mL, about 5.5 pg/mL to about 40.0 pg/mL, about6.0 pg/mL to about 40.0 pg/mL, about 6.5 pg/mL to about 40.0 pg/mL,about 7.0 pg/mL to about 40.0 pg/mL, about 7.5 pg/mL to about 40.0pg/mL, about 8.0 pg/mL to about 40.0 pg/mL, about 8.5 pg/mL to about40.0 pg/mL, about 9.0 pg/mL to about 40.0 pg/mL, about 9.5 pg/mL toabout 40.0 pg/mL, about 10.0 pg/mL to about 40.0 pg/mL, about 1.0 pg/mLto about 35.0 pg/mL, about 1.5 pg/mL to about 35.0 pg/mL, about 2.0pg/mL to about 35.0 pg/mL, about 2.5 pg/mL to about 35.0 pg/mL, about3.0 pg/mL to about 35.0 pg/mL, about 3.5 pg/mL to about 35.0 pg/mL,about 4.0 pg/mL to about 35.0 pg/mL, about 4.5 pg/mL to about 35.0pg/mL, about 5.0 pg/mL to about 35.0 pg/mL, about 5.5 pg/mL to about35.0 pg/mL, about 6.0 pg/mL to about 35.0 pg/mL, about 6.5 pg/mL toabout 35.0 pg/mL, about 7.0 pg/mL to about 35.0 pg/mL, about 7.5 pg/mLto about 35.0 pg/mL, about 8.0 pg/mL to about 35.0 pg/mL, about 8.5pg/mL to about 35.0 pg/mL, about 9.0 pg/mL to about 35.0 pg/mL, about9.5 pg/mL to about 35.0 pg/mL, about 10.0 pg/mL to about 35.0 pg/mL,about 1.0 pg/mL to about 30.0 pg/mL, about 1.5 pg/mL to about 30.0pg/mL, about 2.0 pg/mL to about 30.0 pg/mL, about 2.5 pg/mL to about30.0 pg/mL, about 3.0 pg/mL to about 30.0 pg/mL, about 3.5 pg/mL toabout 30.0 pg/mL, about 4.0 pg/mL to about 30.0 pg/mL, about 4.5 pg/mLto about 30.0 pg/mL, about 5.0 pg/mL to about 30.0 pg/mL, about 5.5pg/mL to about 30.0 pg/mL, about 6.0 pg/mL to about 30.0 pg/mL, about6.5 pg/mL to about 30.0 pg/mL, about 7.0 pg/mL to about 30.0 pg/mL,about 7.5 pg/mL to about 30.0 pg/mL, about 8.0 pg/mL to about 30.0pg/mL, about 8.5 pg/mL to about 30.0 pg/mL, about 9.0 pg/mL to about30.0 pg/mL, about 9.5 pg/mL to about 30.0 pg/mL, about 10.0 pg/mL toabout 30.0 pg/mL, about 1.0 pg/mL to about 29.0 pg/mL, about 1.5 pg/mLto about 29.0 pg/mL, about 2.0 pg/mL to about 29.0 pg/mL, about 2.5pg/mL to about 29.0 pg/mL, about 3.0 pg/mL to about 29.0 pg/mL, about3.5 pg/mL to about 29.0 pg/mL, about 4.0 pg/mL to about 29.0 pg/mL,about 4.5 pg/mL to about 29.0 pg/mL, about 5.0 pg/mL to about 29.0pg/mL, about 5.5 pg/mL to about 29.0 pg/mL, about 6.0 pg/mL to about29.0 pg/mL, about 6.5 pg/mL to about 29.0 pg/mL, about 7.0 pg/mL toabout 29.0 pg/mL, about 7.5 pg/mL to about 29.0 pg/mL, about 8.0 pg/mLto about 29.0 pg/mL, about 8.5 pg/mL to about 29.0 pg/mL, about 9.0pg/mL to about 29.0 pg/mL, about 9.5 pg/mL to about 29.0 pg/mL, about10.0 pg/mL to about 29.0 pg/mL, about 1.0 pg/mL to about 28.0 pg/mL,about 1.5 pg/mL to about 28.0 pg/mL, about 2.0 pg/mL to about 28.0pg/mL, about 2.5 pg/mL to about 28.0 pg/mL, about 3.0 pg/mL to about28.0 pg/mL, about 3.5 pg/mL to about 28.0 pg/mL, about 4.0 pg/mL toabout 28.0 pg/mL, about 4.5 pg/mL to about 28.0 pg/mL, about 5.0 pg/mLto about 28.0 pg/mL, about 5.5 pg/mL to about 28.0 pg/mL, about 6.0pg/mL to about 28.0 pg/mL, about 6.5 pg/mL to about 28.0 pg/mL, about7.0 pg/mL to about 28.0 pg/mL, about 7.5 pg/mL to about 28.0 pg/mL,about 8.0 pg/mL to about 28.0 pg/mL, about 8.5 pg/mL to about 28.0pg/mL, about 9.0 pg/mL to about 28.0 pg/mL, about 9.5 pg/mL to about28.0 pg/mL, about 10.0 pg/mL to about 28.0 pg/mL, about 1.0 pg/mL toabout 27.0 pg/mL, about 1.5 pg/mL to about 27.0 pg/mL, about 2.0 pg/mLto about 27.0 pg/mL, about 2.5 pg/mL to about 27.0 pg/mL, about 3.0pg/mL to about 27.0 pg/mL, about 3.5 pg/mL to about 27.0 pg/mL, about4.0 pg/mL to about 27.0 pg/mL, about 4.5 pg/mL to about 27.0 pg/mL,about 5.0 pg/mL to about 27.0 pg/mL, about 5.5 pg/mL to about 27.0pg/mL, about 6.0 pg/mL to about 27.0 pg/mL, about 6.5 pg/mL to about27.0 pg/mL, about 7.0 pg/mL to about 27.0 pg/mL, about 7.5 pg/mL toabout 27.0 pg/mL, about 8.0 pg/mL to about 27.0 pg/mL, about 8.5 pg/mLto about 27.0 pg/mL, about 9.0 pg/mL to about 27.0 pg/mL, about 9.5pg/mL to about 27.0 pg/mL, about 10.0 pg/mL to about 27.0 pg/mL, about1.0 pg/mL to about 26.0 pg/mL, about 1.5 pg/mL to about 26.0 pg/mL,about 2.0 pg/mL to about 26.0 pg/mL, about 2.5 pg/mL to about 26.0pg/mL, about 3.0 pg/mL to about 26.0 pg/mL, about 3.5 pg/mL to about26.0 pg/mL, about 4.0 pg/mL to about 26.0 pg/mL, about 4.5 pg/mL toabout 26.0 pg/mL, about 5.0 pg/mL to about 26.0 pg/mL, about 5.5 pg/mLto about 26.0 pg/mL, about 6.0 pg/mL to about 26.0 pg/mL, about 6.5pg/mL to about 26.0 pg/mL, about 7.0 pg/mL to about 26.0 pg/mL, about7.5 pg/mL to about 26.0 pg/mL, about 8.0 pg/mL to about 26.0 pg/mL,about 8.5 pg/mL to about 26.0 pg/mL, about 9.0 pg/mL to about 26.0pg/mL, about 9.5 pg/mL to about 26.0 pg/mL, about 10.0 pg/mL to about26.0 pg/mL, about 1.0 pg/mL to about 25.0 pg/mL, about 1.5 pg/mL toabout 25.0 pg/mL, about 2.0 pg/mL to about 25.0 pg/mL, about 2.5 pg/mLto about 25.0 pg/mL, about 3.0 pg/mL to about 25.0 pg/mL, about 3.5pg/mL to about 25.0 pg/mL, about 4.0 pg/mL to about 25.0 pg/mL, about4.5 pg/mL to about 25.0 pg/mL, about 5.0 pg/mL to about 25.0 pg/mL,about 5.5 pg/mL to about 25.0 pg/mL, about 6.0 pg/mL to about 25.0pg/mL, about 6.5 pg/mL to about 25.0 pg/mL, about 7.0 pg/mL to about25.0 pg/mL, about 7.5 pg/mL to about 25.0 pg/mL, about 8.0 pg/mL toabout 25.0 pg/mL, about 8.5 pg/mL to about 25.0 pg/mL, about 9.0 pg/mLto about 25.0 pg/mL, about 9.5 pg/mL to about 25.0 pg/mL, about 10.0pg/mL to about 25.0 pg/mL, about 1.0 pg/mL to about 24.0 pg/mL, about1.5 pg/mL to about 24.0 pg/mL, about 2.0 pg/mL to about 24.0 pg/mL,about 2.5 pg/mL to about 24.0 pg/mL, about 3.0 pg/mL to about 24.0pg/mL, about 3.5 pg/mL to about 24.0 pg/mL, about 4.0 pg/mL to about24.0 pg/mL, about 4.5 pg/mL to about 24.0 pg/mL, about 5.0 pg/mL toabout 24.0 pg/mL, about 5.5 pg/mL to about 24.0 pg/mL, about 6.0 pg/mLto about 24.0 pg/mL, about 6.5 pg/mL to about 24.0 pg/mL, about 7.0pg/mL to about 24.0 pg/mL, about 7.5 pg/mL to about 24.0 pg/mL, about8.0 pg/mL to about 24.0 pg/mL, about 8.5 pg/mL to about 24.0 pg/mL,about 9.0 pg/mL to about 24.0 pg/mL, about 9.5 pg/mL to about 24.0pg/mL, about 10.0 pg/mL to about 24.0 pg/mL, about 1.0 pg/mL to about23.0 pg/mL, about 1.5 pg/mL to about 23.0 pg/mL, about 2.0 pg/mL toabout 23.0 pg/mL, about 2.5 pg/mL to about 23.0 pg/mL, about 3.0 pg/mLto about 23.0 pg/mL, about 3.5 pg/mL to about 23.0 pg/mL, about 4.0pg/mL to about 23.0 pg/mL, about 4.5 pg/mL to about 23.0 pg/mL, about5.0 pg/mL to about 23.0 pg/mL, about 5.5 pg/mL to about 23.0 pg/mL,about 6.0 pg/mL to about 23.0 pg/mL, about 6.5 pg/mL to about 23.0pg/mL, about 7.0 pg/mL to about 23.0 pg/mL, about 7.5 pg/mL to about23.0 pg/mL, about 8.0 pg/mL to about 23.0 pg/mL, about 8.5 pg/mL toabout 23.0 pg/mL, about 9.0 pg/mL to about 23.0 pg/mL, about 9.5 pg/mLto about 23.0 pg/mL, about 10.0 pg/mL to about 23.0 pg/mL, about 1.0pg/mL to about 22.0 pg/mL, about 1.5 pg/mL to about 22.0 pg/mL, about2.0 pg/mL to about 22.0 pg/mL, about 2.5 pg/mL to about 22.0 pg/mL,about 3.0 pg/mL to about 22.0 pg/mL, about 3.5 pg/mL to about 22.0pg/mL, about 4.0 pg/mL to about 22.0 pg/mL, about 4.5 pg/mL to about22.0 pg/mL, about 5.0 pg/mL to about 22.0 pg/mL, about 5.5 pg/mL toabout 22.0 pg/mL, about 6.0 pg/mL to about 22.0 pg/mL, about 6.5 pg/mLto about 22.0 pg/mL, about 7.0 pg/mL to about 22.0 pg/mL, about 7.5pg/mL to about 22.0 pg/mL, about 8.0 pg/mL to about 22.0 pg/mL, about8.5 pg/mL to about 22.0 pg/mL, about 9.0 pg/mL to about 22.0 pg/mL,about 9.5 pg/mL to about 22.0 pg/mL, about 10.0 pg/mL to about 22.0pg/mL, about 1.0 pg/mL to about 21.0 pg/mL, about 1.5 pg/mL to about21.0 pg/mL, about 2.0 pg/mL to about 21.0 pg/mL, about 2.5 pg/mL toabout 21.0 pg/mL, about 3.0 pg/mL to about 21.0 pg/mL, about 3.5 pg/mLto about 21.0 pg/mL, about 4.0 pg/mL to about 21.0 pg/mL, about 4.5pg/mL to about 21.0 pg/mL, about 5.0 pg/mL to about 21.0 pg/mL, about5.5 pg/mL to about 21.0 pg/mL, about 6.0 pg/mL to about 21.0 pg/mL,about 6.5 pg/mL to about 21.0 pg/mL, about 7.0 pg/mL to about 21.0pg/mL, about 7.5 pg/mL to about 21.0 pg/mL, about 8.0 pg/mL to about21.0 pg/mL, about 8.5 pg/mL to about 21.0 pg/mL, about 9.0 pg/mL toabout 21.0 pg/mL, about 9.5 pg/mL to about 21.0 pg/mL, about 10.0 pg/mLto about 21.0 pg/mL, about 1.0 pg/mL to about 20.0 pg/mL, about 1.5pg/mL to about 20.0 pg/mL, about 2.0 pg/mL to about 20.0 pg/mL, about2.5 pg/mL to about 20.0 pg/mL, about 3.0 pg/mL to about 20.0 pg/mL,about 3.5 pg/mL to about 20.0 pg/mL, about 4.0 pg/mL to about 20.0pg/mL, about 4.5 pg/mL to about 20.0 pg/mL, about 5.0 pg/mL to about20.0 pg/mL, about 5.5 pg/mL to about 20.0 pg/mL, about 6.0 pg/mL toabout 20.0 pg/mL, about 6.5 pg/mL to about 20.0 pg/mL, about 7.0 pg/mLto about 20.0 pg/mL, about 7.5 pg/mL to about 20.0 pg/mL, about 8.0pg/mL to about 20.0 pg/mL, about 8.5 pg/mL to about 20.0 pg/mL, about9.0 pg/mL to about 20.0 pg/mL, about 9.5 pg/mL to about 20.0 pg/mL,about 10.0 pg/mL to about 20.0 pg/mL, about 1.0 pg/mL to about 19.0pg/mL, about 1.5 pg/mL to about 19.0 pg/mL, about 2.0 pg/mL to about19.0 pg/mL, about 2.5 pg/mL to about 19.0 pg/mL, about 3.0 pg/mL toabout 19.0 pg/mL, about 3.5 pg/mL to about 19.0 pg/mL, about 4.0 pg/mLto about 19.0 pg/mL, about 4.5 pg/mL to about 19.0 pg/mL, about 5.0pg/mL to about 19.0 pg/mL, about 5.5 pg/mL to about 19.0 pg/mL, about6.0 pg/mL to about 19.0 pg/mL, about 6.5 pg/mL to about 19.0 pg/mL,about 7.0 pg/mL to about 19.0 pg/mL, about 7.5 pg/mL to about 19.0pg/mL, about 8.0 pg/mL to about 19.0 pg/mL, about 8.5 pg/mL to about19.0 pg/mL, about 9.0 pg/mL to about 19.0 pg/mL, about 9.5 pg/mL toabout 19.0 pg/mL, about 10.0 pg/mL to about 19.0 pg/mL, about 1.0 pg/mLto about 18.0 pg/mL, about 1.5 pg/mL to about 18.0 pg/mL, about 2.0pg/mL to about 18.0 pg/mL, about 2.5 pg/mL to about 18.0 pg/mL, about3.0 pg/mL to about 18.0 pg/mL, about 3.5 pg/mL to about 18.0 pg/mL,about 4.0 pg/mL to about 18.0 pg/mL, about 4.5 pg/mL to about 18.0pg/mL, about 5.0 pg/mL to about 18.0 pg/mL, about 5.5 pg/mL to about18.0 pg/mL, about 6.0 pg/mL to about 18.0 pg/mL, about 6.5 pg/mL toabout 18.0 pg/mL, about 7.0 pg/mL to about 18.0 pg/mL, about 7.5 pg/mLto about 18.0 pg/mL, about 8.0 pg/mL to about 18.0 pg/mL, about 8.5pg/mL to about 18.0 pg/mL, about 9.0 pg/mL to about 18.0 pg/mL, about9.5 pg/mL to about 18.0 pg/mL, about 10.0 pg/mL to about 18.0 pg/mL,about 1.0 pg/mL to about 17.0 pg/mL, about 1.5 pg/mL to about 17.0pg/mL, about 2.0 pg/mL to about 17.0 pg/mL, about 2.5 pg/mL to about17.0 pg/mL, about 3.0 pg/mL to about 17.0 pg/mL, about 3.5 pg/mL toabout 17.0 pg/mL, about 4.0 pg/mL to about 17.0 pg/mL, about 4.5 pg/mLto about 17.0 pg/mL, about 5.0 pg/mL to about 17.0 pg/mL, about 5.5pg/mL to about 17.0 pg/mL, about 6.0 pg/mL to about 17.0 pg/mL, about6.5 pg/mL to about 17.0 pg/mL, about 7.0 pg/mL to about 17.0 pg/mL,about 7.5 pg/mL to about 17.0 pg/mL, about 8.0 pg/mL to about 17.0pg/mL, about 8.5 pg/mL to about 17.0 pg/mL, about 9.0 pg/mL to about17.0 pg/mL, about 9.5 pg/mL to about 17.0 pg/mL, about 10.0 pg/mL toabout 17.0 pg/mL, about 1.0 pg/mL to about 16.0 pg/mL, about 1.5 pg/mLto about 16.0 pg/mL, about 2.0 pg/mL to about 16.0 pg/mL, about 2.5pg/mL to about 16.0 pg/mL, about 3.0 pg/mL to about 16.0 pg/mL, about3.5 pg/mL to about 16.0 pg/mL, about 4.0 pg/mL to about 16.0 pg/mL,about 4.5 pg/mL to about 16.0 pg/mL, about 5.0 pg/mL to about 16.0pg/mL, about 5.5 pg/mL to about 16.0 pg/mL, about 6.0 pg/mL to about16.0 pg/mL, about 6.5 pg/mL to about 16.0 pg/mL, about 7.0 pg/mL toabout 16.0 pg/mL, about 7.5 pg/mL to about 16.0 pg/mL, about 8.0 pg/mLto about 16.0 pg/mL, about 8.5 pg/mL to about 16.0 pg/mL, about 9.0pg/mL to about 16.0 pg/mL, about 9.5 pg/mL to about 16.0 pg/mL, about10.0 pg/mL to about 16.0 pg/mL, about 1.0 pg/mL to about 15.0 pg/mL,about 1.5 pg/mL to about 15.0 pg/mL, about 2.0 pg/mL to about 15.0pg/mL, about 2.5 pg/mL to about 15.0 pg/mL, about 3.0 pg/mL to about15.0 pg/mL, about 3.5 pg/mL to about 15.0 pg/mL, about 4.0 pg/mL toabout 15.0 pg/mL, about 4.5 pg/mL to about 15.0 pg/mL, about 5.0 pg/mLto about 15.0 pg/mL, about 5.5 pg/mL to about 15.0 pg/mL, about 6.0pg/mL to about 15.0 pg/mL, about 6.5 pg/mL to about 15.0 pg/mL, about7.0 pg/mL to about 15.0 pg/mL, about 7.5 pg/mL to about 15.0 pg/mL,about 8.0 pg/mL to about 15.0 pg/mL, about 8.5 pg/mL to about 15.0pg/mL, about 9.0 pg/mL to about 15.0 pg/mL, about 9.5 pg/mL to about15.0 pg/mL, or about 10.0 pg/mL to about 15.0 pg/mL.

In some embodiments, the absolute amount of cTnI is determined by anassay having a sensitivity of between at least about 65% to about 100%and a specificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 65% to about100%, between at least about 65% to at least about 99%, between at leastabout 65% to at least about 95%, between at least about 65% to at leastabout 90%, between at least about 65% to at least about 85%, between atleast about 65% to at least about 80%, between at least about 65% to atleast about 75%, between at least about 65% to at least about 70%,between at least about 75% to about 100%, between at least about 75% toat least about 99%, between at least about 75% to at least about 95%,between at least about 75% to at least about 90%, between at least about75% to at least about 85%, between at least about 75% to at least about80%, between at least about 85% to about 100%, between at least about85% to at least about 99%, between at least about 85% to at least about95%, between at least about 85% to at least about 90%, between at leastabout 95% to about 100%, or between at least about 95% to at least about99%. In some embodiments, the sensitivity is at least about 65.0%, atleast about 70.0%, at least about 75.0%, at least about 80.0%, at leastabout 82.4%, at least about 85.0%, at least about 87.5%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 69.5%, at leastabout 70.0%, at least about 75.0%, at least about 80.0%, at least about85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%,at least about 93.0%, at least about 94.0%, at least about 95.0%, atleast about 96.0%, at least about 97.0%, at least about 98.0%, at leastabout 99.0%, at least about 99.1%, at least about 99.2%, at least about99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%,at least about 99.7%, at least about 99.8%, at least about 99.9%, or atleast about 100.0%. For example, the sensitivity is at least about 82.4%and the specificity is at least about 69.5%, the sensitivity is at leastabout 99% and the specificity is at least about 75%, the sensitivity isat least about 99% and the specificity is at least about 99%, or thesensitivity is at least about 100% and the specificity is at least about100%.

In some embodiments, the absolute amount can be between at least about 1pg/mL to about 25 pg/mL. In some embodiments, the absolute amount can bebetween at least about 1 pg/mL to about 25 pg/mL, between at least about1 pg/mL to about 20 pg/mL, between at least about 1 pg/mL to about 15pg/mL, between at least about 1 pg/mL to about 10 pg/mL, between atleast about 1 pg/mL to about 9 pg/mL, between at least about 1 pg/mL toabout 8 pg/mL, between at least about 1 pg/mL to about 7 pg/mL, betweenat least about 1 pg/mL to about 6 pg/mL, between at least about 1 pg/mLto about 5 pg/mL, between at least about 1 pg/mL to about 4 pg/mL,between at least about 1 pg/mL to about 3 pg/mL, between at least about1 pg/mL to about 2 pg/mL, between at least about 1 pg/mL to about 1.5pg/mL, between at least about 1.5 pg/mL to about 25 pg/mL, between atleast about 1.5 pg/mL to about 20 pg/mL, between at least about 1.5pg/mL to about 15 pg/mL, between at least about 1.5 pg/mL to about 10pg/mL, between at least about 1.5 pg/mL to about 9 pg/mL, between atleast about 1.5 pg/mL to about 8 pg/mL, between at least about 1.5 pg/mLto about 7 pg/mL, between at least about 1.5 pg/mL to about 6 pg/mL,between at least about 1.5 pg/mL to about 5 pg/mL, between at leastabout 1.5 pg/mL to about 4 pg/mL, between at least about 1.5 pg/mL toabout 3 pg/mL, between at least about 1.5 pg/mL to about 2 pg/mL,between at least about 2 pg/mL to about 25 pg/mL, between at least about2 pg/mL to about 20 pg/mL, between at least about 2 pg/mL to about 15pg/mL, between at least about 2 pg/mL to about 10 pg/mL, between atleast about 2 pg/mL to about 9 pg/mL, between at least about 2 pg/mL toabout 8 pg/mL, between at least about 2 pg/mL to about 7 pg/mL, betweenat least about 2 pg/mL to about 6 pg/mL, between at least about 2 pg/mLto about 5 pg/mL, between at least about 2 pg/mL to about 4 pg/mL,between at least about 2 pg/mL to about 3 pg/mL, between at least about3 pg/mL to about 25 pg/mL, between at least about 3 pg/mL to about 20pg/mL, between at least about 3 pg/mL to about 15 pg/mL, between atleast about 3 pg/mL to about 10 pg/mL, between at least about 3 pg/mL toabout 9 pg/mL, between at least about 3 pg/mL to about 8 pg/mL, betweenat least about 3 pg/mL to about 7 pg/mL, between at least about 3 pg/mLto about 6 pg/mL, between at least about 3 pg/mL to about 5 pg/mL,between at least about 3 pg/mL to about 4 pg/mL, between at least about4 pg/mL to about 25 pg/mL, between at least about 4 pg/mL to about 20pg/mL, between at least about 4 pg/mL to about 15 pg/mL, between atleast about 4 pg/mL to about 10 pg/mL, between at least about 4 pg/mL toabout 9 pg/mL, between at least about 4 pg/mL to about 8 pg/mL, betweenat least about 4 pg/mL to about 7 pg/mL, between at least about 4 pg/mLto about 6 pg/mL, between at least about 4 pg/mL to about 5 pg/mL,between at least about 5 pg/mL to about 25 pg/mL, between at least about5 pg/mL to about 20 pg/mL, between at least about 5 pg/mL to about 15pg/mL, between at least about 5 pg/mL to about 10 pg/mL, between atleast about 5 pg/mL to about 9 pg/mL, between at least about 5 pg/mL toabout 8 pg/mL, between at least about 5 pg/mL to about 7 pg/mL, betweenat least about 5 pg/mL to about 6 pg/mL, between at least about 6 pg/mLto about 25 pg/mL, between at least about 6 pg/mL to about 20 pg/mL,between at least about 6 pg/mL to about 15 pg/mL, between at least about6 pg/mL to about 10 pg/mL, between at least about 6 pg/mL to about 9pg/mL, between at least about 6 pg/mL to about 8 pg/mL, between at leastabout 6 pg/mL to about 7 pg/mL, between at least about 7 pg/mL to about25 pg/mL, between at least about 7 pg/mL to about 20 pg/mL, between atleast about 7 pg/mL to about 15 pg/mL, between at least about 7 pg/mL toabout 10 pg/mL, between at least about 7 pg/mL to about 9 pg/mL, betweenat least about 7 pg/mL to about 8 pg/mL, between at least about 8 pg/mLto about 25 pg/mL, between at least about 8 pg/mL to about 20 pg/mL,between at least about 8 pg/mL to about 15 pg/mL, between at least about8 pg/mL to about 10 pg/mL, between at least about 8 pg/mL to about 9pg/mL, between at least about 9 pg/mL to about 25 pg/mL, between atleast about 9 pg/mL to about 20 pg/mL, between at least about 9 pg/mL toabout 15 pg/mL, between at least about 9 pg/mL to about 10 pg/mL,between at least about 10 pg/mL to about 25 pg/mL, between at leastabout 10 pg/mL to about 20 pg/mL, between at least about 10 pg/mL toabout 15 pg/mL, between at least about 15 pg/mL to about 25 pg/mL,between at least about 15 pg/mL to about 20 pg/mL, or between at leastabout 20 pg/mL to about 25 pg/mL. In some embodiments, the absoluteamount can be at least about 0.5 pg/mL, at least about 1 pg/mL, at leastabout 1.5 pg/mL, at least about 2 pg/mL, at least about 3 pg/mL, atleast about 4 pg/mL, at least about 5 pg/mL, at least about 6 pg/mL, atleast about 7 pg/mL, at least about 8 pg/mL, at least about 9 pg/mL, atleast about 10 pg/mL, at least about 11 pg/mL, at least about 12 pg/mL,at least about 13 pg/mL, at least about 14 pg/mL, at least about 15pg/mL, at least about 20 pg/mL, or at least about 25 pg/mL.

In some embodiments, the absolute amount is between about 1 pg/mL toabout 50 pg/mL, between about 1 pg/mL to about 40 pg/mL, between about 1pg/mL to about 30 pg/mL, between about 1 pg/mL to about 20 pg/mL,between about 1 pg/mL to about 10 pg/mL, between about 1 pg/mL to about5 pg/mL, between about 2.5 pg/mL to about 50 pg/mL, between about 2.5pg/mL to about 40 pg/mL, between about 2.5 pg/mL to about 30 pg/mL,between about 2.5 pg/mL to about 20 pg/mL, between about 2.5 pg/mL toabout 10 pg/mL, between about 2.5 pg/mL to about 5 pg/mL, between about5 pg/mL to about 50 pg/mL, between about 5 pg/mL to about 40 pg/mL,between about 5 pg/mL to about 30 pg/mL, between about 5 pg/mL to about20 pg/mL, or between about 5 pg/mL to about 10 pg/mL. In someembodiments, the absolute amount is at least about 1 pg/mL, at leastabout 2 pg/mL, at least about 3 pg/mL, at least about 4 pg/mL, at leastabout 5 pg/mL, at least about 5.5 pg/mL, at least about 5.6 pg/mL, atleast about 5.7 pg/mL at least about 5.8 pg/mL at least about 5.9 pg/mL,at least about 6 pg/mL, at least about 7 pg/mL, at least about 8 pg/mL,at least about 9 pg/mL, at least about 10 pg/mL, at least about 20pg/mL, at least about 30 pg/mL, at least about 40 pg/mL, or at leastabout 10 pg/mL.

In some embodiments, the method further includes treating the humansubject predicted as having an unfavorable outcome with a traumaticbrain injury treatment, as described below. In some embodiments, themethod further includes monitoring the human subject predicted as havingan unfavorable outcome, as described below. In some embodiments, themethod further includes ordering additional tests to obtain furtherclinical information about the mild TBI in the human subject predictedas having an unfavorable outcome. In some embodiments, the methodincludes treating the human subject predicted to have an unfavorableoutcome with a cardioprotective treatment to protect the heart asdescribed below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assaysare described in further detail herein in Sections 11-13. Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. For example, a clinical chemistryformat can include an assay that involves one antibody or no antibody.Examples of analyzers that can be used for the clinical chemistry formatare described in U.S. Patent publication Nos. 2016/0320422 and2015/0112630.

8. Methods of Aiding in Predicting or Predicting the Outcome of a HumanSubject Having Mild Traumatic Brain Injury Using Cardiac Troponin I(cTnI) and Age

The present disclosure relates, among other methods, to a method ofaiding in predicting (or predicting) the outcome of a human subjecthaving mild traumatic brain injury (TBI), e.g., determining whether thesubject will have an unfavorable or negative outcome or favorableoutcome. As used herein, the phrase “determining whether the subject hasa favorable outcome” refers to the fact that the aforementioned methodcan be used, e.g., with other information (e.g., clinical assessmentdata), to determine that the subject is more likely than not to have apositive outcome from the mild TBI. Additionally, as used herein, thephrase “determining whether the subject has an unfavorable outcome”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have an unfavorable ornegative outcome from the mild TBI.

As mentioned above, the methods described herein can be used todetermine whether a subject diagnosed with a mild TBI is more likelythan not to have (1) a favorable outcome (optionally, the favorableoutcome can be that the subject fully recovers and does not continue toexperience one or more symptoms of a mild TBI); or (2) an unfavorableoutcome (optionally, the unfavorable outcome can be that the subjectdoes not fully recover and does continue to experience one or moresymptoms of a mild TBI).

Alternatively and optionally, a favorable outcome can mean that thesubject is more likely than not to suffer no more than onepost-concussion syndrome symptom as a result of the mild TBI such as:(a) physical difficulties (e.g., headaches, dizziness, fatigue,sensitivity to light noise and light, etc.); (b) cognitive difficulties(e.g., trouble concentration, memory problems, restlessness, etc.); (c)emotional difficulties (e.g., personality changes, irritability,depression, apathy, etc.); or (d) sleep difficulties (e.g., insomnia,etc.). Alternatively and option, subject who have an unfavorable outcomeare more likely to suffer from more than one post-concussion syndromesymptom such as: (a) physical difficulties (e.g., headaches, dizziness,fatigue, sensitivity to light noise and light, etc.); (b) cognitivedifficulties (e.g., trouble concentration, memory problems,restlessness, etc.); (c) emotional difficulties (e.g, personalitychanges, irritability, depression, apathy, etc.); (d) sleep difficulties(e.g., insomnia, etc.); or (e) any combinations of (a)-(d)).Alternatively and optionally, an unfavorable outcome can also mean thata subject exhibits one or more symptoms of mild TBI. Alternatively andoptionally, an unfavorable outcome can also mean that the subject'sconditions worsens from mild TBI to moderate, moderate to severe orsevere. Additionally, subjects having a favorable outcome are likely tohave a GOSE score of 5 or greater whereas subjects having an unfavorableoutcome are likely to have a GOSE score of less than 5.

The methods disclosed herein can include performing an assay on a firstsample and second sample obtained from the human subject after an injuryto the head and determining the age of the subject. The first sample istaken from the subject within about 24 hours after injury and the secondsample is taken from the subject within 0 to about 4 hours after thefirst sample is taken at two or more different time points. The assay isused to determine the level of cardiac troponin I in each of thesesamples. If the amount or level of cardiac troponin I in the firstsample and/or second sample is higher than a reference level of cardiactroponin I and the age of the subject is lower than a reference age,such as, for example, between 40 and 50 years of age, then adetermination is made that the subject is predicted to have anunfavorable outcome, for example at 6 months, or a more severe traumaticbrain injury. However, if the amount or level of cardiac troponin I inthe first sample and the second sample is lower than the reference levelof cardiac troponin I and the age of the subject is less than areference age, then a determination is made that the subject ispredicted to have a favorable outcome at 6 months or a less severetraumatic brain injury.

In some embodiments of the above method, the age of the subject is 18 to30 years. In other embodiments of the above method, the age of thesubject is 31 to 50 years. In yet other embodiments of the above method,the age of the subject is 51 to 70 years. In yet other embodiments ofthe above method, the age of the subject is 70 to 100 years. In yetother embodiments of the above method, the age is 18 years or less. Inyet other embodiments of the above method, the age is 19-50 years. Instill further embodiments, the age is 51-70 years. In yet otherembodiments of the above method, the age is greater than 70 years. Inyet other embodiments of the above method, the age of the subject is 20to 30 years. In yet other embodiments of the above method, the age ofthe subject is 31 to 40 years. In yet other embodiments of the abovemethod, the age of the subject is 41 to 50 years. In yet otherembodiments of the above method, the age of the subject is 51 to 60years. In yet other embodiments of the above method, the age of thesubject is 61 to 70 years. In yet other embodiments of the above method,the age of the subject is 71 to 80 years. In yet other embodiments ofthe above method, the age of the subject is 81 to 90 years. In yet otherembodiments of the above method, the age of the subject is 91 to 100years.

In some embodiments, the cTnI levels in samples taken at a first timepoint and at a second time point are combined with the age of thesubject in the following formula:

${\Pr\left( {Y = 1} \right)} = \frac{\exp\begin{pmatrix}{\beta_{0} + {\beta_{1}{\ln\left( {{initial}\mspace{14mu}{troponin}} \right)}} +} \\{{\beta_{2}{\ln\left( {4\mspace{14mu}{hour}\mspace{14mu}{troponin}} \right)}} + {\beta_{3}{age}}}\end{pmatrix}}{1 + {\exp\begin{pmatrix}{\beta_{0} + {\beta_{1}{\ln\left( {{initial}\mspace{14mu}{troponin}} \right)}} +} \\{{\beta_{2}{\ln\left( {4\mspace{14mu}{hour}\mspace{14mu}{troponin}} \right)}} + {\beta_{3}{age}}}\end{pmatrix}}}$

In some embodiments, age is a continuous variable. For examples, ifweights are assigned based on the co-efficients, the weight of age canbe 1.06, the weight of initial troponin can be 0.25, and the weight of 4hour troponin can be 4.22. The first (or previous) sample may beobtained or taken from the subject 0 minutes, within about 1 minute,within about 2 minutes, within about 3 minutes, within about 4 minutes,within about 5 minutes, within about 6 minutes, within about 7 minutes,within about 8 minutes, within about 9 minutes, within about 10 minutes,within about 11 minutes, within about 12 minutes, within about 13minutes, within about 14 minutes, within about 15 minutes, within about20 minutes, within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours or withinabout 24 hours of an injury to the head. In some embodiments, the (orprevious) sample is taken within about 0 to about 1 hours, within about0 to about 2 hours, within about 0 to about 3 hours, within about 0 toabout 4 hours, within about 0 to about 5 hours, within about 0 to about6 hours, within about 0 to about 7 hours, within about 0 to about 8hours, within about 0 to about 9 hours, within about 0 to about 10hours, within about 0 to about 11 hours, within about 0 to about 12hours, within about 0 to about 18 hours, within about 6 to about 12hours, within about 12 to about 18 hours, within about 18 to about 24hours, or greater than 24 hours after the injury. The second (orsubsequent) sample can be obtained after about 0 hour to 6 hours afterthe first (or last) sample was obtained. In some embodiments, the second(or subsequent) sample is taken from the subject within at least about30 minutes, within at least about 1 hour, within at least about 2 hours,within at least about 3 hours, within at least about 4 hours, within atleast about 5 hours, or within at least about 6 hours after the firstsample is taken from the subject.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cardiac troponin is determined at oneor more time points. In certain embodiments, the subject may besuspected of having a mild traumatic brain injury based on the GlasgowComa Scale score. In certain embodiments, the subject may be suspectedof having a mild traumatic brain injury based on an abnormal head CT. Insome embodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the subject has received a GOSE score after theassay is performed. In some embodiments, the subject is suspected ashaving an unfavorable outcome based on the GOSE score. In someembodiments, the subject has a GOSE score of less than 5 at 1 month, 2months, 3 months, 4 months, 5 months, or 6 months after the injury. Insome embodiments, the reference level of cTnI is correlated withsubjects having an unfavorable outcome. In some embodiments, thereference level of cTnI is correlated with a GOSE score of 1-5. In someembodiments, the subject is suspected as having a favorable outcomebased on the GOSE score. In some embodiments, the reference level ofcTnI is correlated with subjects having a favorable outcome. In someembodiments, the reference level of cTnI is correlated with a GOSE scoreof 6-8.

In some embodiments, the reference level of cTnI is correlated withsubjects having a more severe traumatic brain injury, such as moderateto severe traumatic brain injury. In some embodiments, the referencelevel of cTnI is correlated with a Glasgow Coma Scale score of 3-12. Insome embodiments, the subject is suspected as having mild traumaticbrain injury based on the Glasgow Coma Scale score. In some embodiments,the reference level of cTnI is correlated with subjects having a lesssevere traumatic brain injury, such as mild traumatic brain injury. Insome embodiments, the reference level of cTnI is correlated with aGlasgow Coma Scale score of 13-15.

Generally, a reference level of cTnI can also be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In certain embodiments, the reference level may be correlated withcontrol subjects that have not sustained a head injury.

In some embodiments, the method can include obtaining samples from thesubject and contacting the samples with an antibody for cardiac troponinI to allow formation of a complex of the antibody and cardiac troponinI. The method also includes detecting the resulting antibody-cardiactroponin I complex.

In some embodiments, the reference level of cTnI is determined by anassay having a sensitivity of between at least about 65% to about 100%and a specificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 65% to about100%, between at least about 65% to at least about 99%, between at leastabout 65% to at least about 95%, between at least about 65% to at leastabout 90%, between at least about 65% to at least about 85%, between atleast about 65% to at least about 80%, between at least about 65% to atleast about 75%, between at least about 65% to at least about 70%,between at least about 75% to about 100%, between at least about 75% toat least about 99%, between at least about 75% to at least about 95%,between at least about 75% to at least about 90%, between at least about75% to at least about 85%, between at least about 75% to at least about80%, between at least about 85% to about 100%, between at least about85% to at least about 99%, between at least about 85% to at least about95%, between at least about 85% to at least about 90%, between at leastabout 95% to about 100%, or between at least about 95% to at least about99%. In some embodiments, the sensitivity is at least about 65.0%, atleast about 70.0%, at least about 75.0%, at least about 80.0%, at leastabout 85.0%, at least about 87.5%, at least about 90.0%, at least about95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%,at least about 99.3%, at least about 99.4%, at least about 99.5%, atleast about 99.6%, at least about 99.7%, at least about 99.8%, at leastabout 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the reference level of cardiac troponin I is fromabout 0.0100 pg/mL to about 50.0 pg/mL, about 0.0200 pg/mL to about 50.0pg/mL, about 0.0300 pg/mL to about 50.0 pg/mL, about 0.036 pg/mL toabout 50.0 pg/mL, about 0.0400 pg/mL to about 50.0 pg/mL, about 0.0500pg/mL to about 50.0 pg/mL, about 0.1 pg/mL to about 50.0 pg/mL, about0.5 pg/mL to about 50.0 pg/mL, about 1.0 pg/mL to about 50.0 pg/mL,about 1.5 pg/mL to about 50.0 pg/mL, about 2.0 pg/mL to about 50.0pg/mL, about 2.5 pg/mL to about 50.0 pg/mL, about 3.0 pg/mL to about50.0 pg/mL, about 3.5 pg/mL to about 50.0 pg/mL, about 4.0 pg/mL toabout 50.0 pg/mL, about 4.5 pg/mL to about 50.0 pg/mL, about 5.0 pg/mLto about 50.0 pg/mL, about 5.5 pg/mL to about 50.0 pg/mL, about 6.0pg/mL to about 50.0 pg/mL, about 6.5 pg/mL to about 50.0 pg/mL, about7.0 pg/mL to about 50.0 pg/mL, about 7.5 pg/mL to about 50.0 pg/mL,about 8.0 pg/mL to about 50.0 pg/mL, about 8.5 pg/mL to about 50.0pg/mL, about 9.0 pg/mL to about 50.0 pg/mL, about 9.5 pg/mL to about50.0 pg/mL, about 10.0 pg/mL to about 50.0 pg/mL, about 0.0100 pg/mL toabout 40.0 pg/mL, about 0.0200 pg/mL to about 40.0 pg/mL, about 0.0300pg/mL to about 40.0 pg/mL, about 0.036 pg/mL to about 40.0 pg/mL, about0.0400 pg/mL to about 40.0 pg/mL, about 0.0500 pg/mL to about 40.0pg/mL, about 0.1 pg/mL to about 40.0 pg/mL, about 0.5 pg/mL to about40.0 pg/mL, about 1.0 pg/mL to about 40.0 pg/mL, about 1.5 pg/mL toabout 40.0 pg/mL, about 2.0 pg/mL to about 40.0 pg/mL, about 2.5 pg/mLto about 40.0 pg/mL, about 3.0 pg/mL to about 40.0 pg/mL, about 3.5pg/mL to about 40.0 pg/mL, about 4.0 pg/mL to about 40.0 pg/mL, about4.5 pg/mL to about 40.0 pg/mL, about 5.0 pg/mL to about 40.0 pg/mL,about 5.5 pg/mL to about 40.0 pg/mL, about 6.0 pg/mL to about 40.0pg/mL, about 6.5 pg/mL to about 40.0 pg/mL, about 7.0 pg/mL to about40.0 pg/mL, about 7.5 pg/mL to about 40.0 pg/mL, about 8.0 pg/mL toabout 40.0 pg/mL, about 8.5 pg/mL to about 40.0 pg/mL, about 9.0 pg/mLto about 40.0 pg/mL, about 9.5 pg/mL to about 40.0 pg/mL, about 10.0pg/mL to about 40.0 pg/mL, about 0.0100 pg/mL to about 35.0 pg/mL, about0.0200 pg/mL to about 35.0 pg/mL, about 0.0300 pg/mL to about 35.0pg/mL, about 0.036 pg/mL to about 35.0 pg/mL, about 0.0400 pg/mL toabout 35.0 pg/mL, about 0.0500 pg/mL to about 35.0 pg/mL, about 0.1pg/mL to about 35.0 pg/mL, about 0.5 pg/mL to about 35.0 pg/mL, about1.0 pg/mL to about 35.0 pg/mL, about 1.5 pg/mL to about 35.0 pg/mL,about 2.0 pg/mL to about 35.0 pg/mL, about 2.5 pg/mL to about 35.0pg/mL, about 3.0 pg/mL to about 35.0 pg/mL, about 3.5 pg/mL to about35.0 pg/mL, about 4.0 pg/mL to about 35.0 pg/mL, about 4.5 pg/mL toabout 35.0 pg/mL, about 5.0 pg/mL to about 35.0 pg/mL, about 5.5 pg/mLto about 35.0 pg/mL, about 6.0 pg/mL to about 35.0 pg/mL, about 6.5pg/mL to about 35.0 pg/mL, about 7.0 pg/mL to about 35.0 pg/mL, about7.5 pg/mL to about 35.0 pg/mL, about 8.0 pg/mL to about 35.0 pg/mL,about 8.5 pg/mL to about 35.0 pg/mL, about 9.0 pg/mL to about 35.0pg/mL, about 9.5 pg/mL to about 35.0 pg/mL, about 10.0 pg/mL to about35.0 pg/mL, about 0.0100 pg/mL to about 30.0 pg/mL, about 0.0200 pg/mLto about 30.0 pg/mL, about 0.0300 pg/mL to about 30.0 pg/mL, about 0.036pg/mL to about 30.0 pg/mL, about 0.0400 pg/mL to about 30.0 pg/mL, about0.0500 pg/mL to about 30.0 pg/mL, about 0.1 pg/mL to about 30.0 pg/mL,about 0.5 pg/mL to about 30.0 pg/mL, about 1.0 pg/mL to about 30.0pg/mL, about 1.5 pg/mL to about 30.0 pg/mL, about 2.0 pg/mL to about30.0 pg/mL, about 2.5 pg/mL to about 30.0 pg/mL, about 3.0 pg/mL toabout 30.0 pg/mL, about 3.5 pg/mL to about 30.0 pg/mL, about 4.0 pg/mLto about 30.0 pg/mL, about 4.5 pg/mL to about 30.0 pg/mL, about 5.0pg/mL to about 30.0 pg/mL, about 5.5 pg/mL to about 30.0 pg/mL, about6.0 pg/mL to about 30.0 pg/mL, about 6.5 pg/mL to about 30.0 pg/mL,about 7.0 pg/mL to about 30.0 pg/mL, about 7.5 pg/mL to about 30.0pg/mL, about 8.0 pg/mL to about 30.0 pg/mL, about 8.5 pg/mL to about30.0 pg/mL, about 9.0 pg/mL to about 30.0 pg/mL, about 9.5 pg/mL toabout 30.0 pg/mL, about 10.0 pg/mL to about 30.0 pg/mL, about 0.0100pg/mL to about 25.0 pg/mL, about 0.0200 pg/mL to about 25.0 pg/mL, about0.0300 pg/mL to about 25.0 pg/mL, about 0.036 pg/mL to about 25.0 pg/mL,about 0.0400 pg/mL to about 25.0 pg/mL, about 0.0500 pg/mL to about 25.0pg/mL, about 0.1 pg/mL to about 25.0 pg/mL, about 0.5 pg/mL to about25.0 pg/mL, about 1.0 pg/mL to about 25.0 pg/mL, about 1.5 pg/mL toabout 25.0 pg/mL, about 2.0 pg/mL to about 25.0 pg/mL, about 2.5 pg/mLto about 25.0 pg/mL, about 3.0 pg/mL to about 25.0 pg/mL, about 3.5pg/mL to about 25.0 pg/mL, about 4.0 pg/mL to about 25.0 pg/mL, about4.5 pg/mL to about 25.0 pg/mL, about 5.0 pg/mL to about 25.0 pg/mL,about 5.5 pg/mL to about 25.0 pg/mL, about 6.0 pg/mL to about 25.0pg/mL, about 6.5 pg/mL to about 25.0 pg/mL, about 7.0 pg/mL to about25.0 pg/mL, about 7.5 pg/mL to about 25.0 pg/mL, about 8.0 pg/mL toabout 25.0 pg/mL, about 8.5 pg/mL to about 25.0 pg/mL, about 9.0 pg/mLto about 25.0 pg/mL, about 9.5 pg/mL to about 25.0 pg/mL, about 10.0pg/mL to about 25.0 pg/mL, about 0.0100 pg/mL to about 24.0 pg/mL, about0.0200 pg/mL to about 24.0 pg/mL, about 0.0300 pg/mL to about 24.0pg/mL, about 0.036 pg/mL to about 24.0 pg/mL, about 0.0400 pg/mL toabout 24.0 pg/mL, about 0.0500 pg/mL to about 24.0 pg/mL, about 0.1pg/mL to about 24.0 pg/mL, about 0.5 pg/mL to about 24.0 pg/mL, about1.0 pg/mL to about 24.0 pg/mL, about 1.5 pg/mL to about 24.0 pg/mL,about 2.0 pg/mL to about 24.0 pg/mL, about 2.5 pg/mL to about 24.0pg/mL, about 3.0 pg/mL to about 24.0 pg/mL, about 3.5 pg/mL to about24.0 pg/mL, about 4.0 pg/mL to about 24.0 pg/mL, about 4.5 pg/mL toabout 24.0 pg/mL, about 5.0 pg/mL to about 24.0 pg/mL, about 5.5 pg/mLto about 24.0 pg/mL, about 6.0 pg/mL to about 24.0 pg/mL, about 6.5pg/mL to about 24.0 pg/mL, about 7.0 pg/mL to about 24.0 pg/mL, about7.5 pg/mL to about 24.0 pg/mL, about 8.0 pg/mL to about 24.0 pg/mL,about 8.5 pg/mL to about 24.0 pg/mL, about 9.0 pg/mL to about 24.0pg/mL, about 9.5 pg/mL to about 24.0 pg/mL, about 10.0 pg/mL to about24.0 pg/mL, about 0.0100 pg/mL to about 23.0 pg/mL, about 0.0200 pg/mLto about 23.0 pg/mL, about 0.0300 pg/mL to about 23.0 pg/mL, about 0.036pg/mL to about 23.0 pg/mL, about 0.0400 pg/mL to about 23.0 pg/mL, about0.0500 pg/mL to about 23.0 pg/mL, about 0.1 pg/mL to about 23.0 pg/mL,about 0.5 pg/mL to about 23.0 pg/mL, about 1.0 pg/mL to about 23.0pg/mL, about 1.5 pg/mL to about 23.0 pg/mL, about 2.0 pg/mL to about23.0 pg/mL, about 2.5 pg/mL to about 23.0 pg/mL, about 3.0 pg/mL toabout 23.0 pg/mL, about 3.5 pg/mL to about 23.0 pg/mL, about 4.0 pg/mLto about 23.0 pg/mL, about 4.5 pg/mL to about 23.0 pg/mL, about 5.0pg/mL to about 23.0 pg/mL, about 5.5 pg/mL to about 23.0 pg/mL, about6.0 pg/mL to about 23.0 pg/mL, about 6.5 pg/mL to about 23.0 pg/mL,about 7.0 pg/mL to about 23.0 pg/mL, about 7.5 pg/mL to about 23.0pg/mL, about 8.0 pg/mL to about 23.0 pg/mL, about 8.5 pg/mL to about23.0 pg/mL, about 9.0 pg/mL to about 23.0 pg/mL, about 9.5 pg/mL toabout 23.0 pg/mL, about 10.0 pg/mL to about 23.0 pg/mL, about 0.0100pg/mL to about 22.0 pg/mL, about 0.0200 pg/mL to about 22.0 pg/mL, about0.0300 pg/mL to about 22.0 pg/mL, about 0.036 pg/mL to about 22.0 pg/mL,about 0.0400 pg/mL to about 22.0 pg/mL, about 0.0500 pg/mL to about 22.0pg/mL, about 0.1 pg/mL to about 22.0 pg/mL, about 0.5 pg/mL to about22.0 pg/mL, about 1.0 pg/mL to about 22.0 pg/mL, about 1.5 pg/mL toabout 22.0 pg/mL, about 2.0 pg/mL to about 22.0 pg/mL, about 2.5 pg/mLto about 22.0 pg/mL, about 3.0 pg/mL to about 22.0 pg/mL, about 3.5pg/mL to about 22.0 pg/mL, about 4.0 pg/mL to about 22.0 pg/mL, about4.5 pg/mL to about 22.0 pg/mL, about 5.0 pg/mL to about 22.0 pg/mL,about 5.5 pg/mL to about 22.0 pg/mL, about 6.0 pg/mL to about 22.0pg/mL, about 6.5 pg/mL to about 22.0 pg/mL, about 7.0 pg/mL to about22.0 pg/mL, about 7.5 pg/mL to about 22.0 pg/mL, about 8.0 pg/mL toabout 22.0 pg/mL, about 8.5 pg/mL to about 22.0 pg/mL, about 9.0 pg/mLto about 22.0 pg/mL, about 9.5 pg/mL to about 22.0 pg/mL, about 10.0pg/mL to about 22.0 pg/mL, about 0.0100 pg/mL to about 21.0 pg/mL, about0.0200 pg/mL to about 21.0 pg/mL, about 0.0300 pg/mL to about 21.0pg/mL, about 0.036 pg/mL to about 21.0 pg/mL, about 0.0400 pg/mL toabout 21.0 pg/mL, about 0.0500 pg/mL to about 21.0 pg/mL, about 0.1pg/mL to about 21.0 pg/mL, about 0.5 pg/mL to about 21.0 pg/mL, about1.0 pg/mL to about 21.0 pg/mL, about 1.5 pg/mL to about 21.0 pg/mL,about 2.0 pg/mL to about 21.0 pg/mL, about 2.5 pg/mL to about 21.0pg/mL, about 3.0 pg/mL to about 21.0 pg/mL, about 3.5 pg/mL to about21.0 pg/mL, about 4.0 pg/mL to about 21.0 pg/mL, about 4.5 pg/mL toabout 21.0 pg/mL, about 5.0 pg/mL to about 21.0 pg/mL, about 5.5 pg/mLto about 21.0 pg/mL, about 6.0 pg/mL to about 21.0 pg/mL, about 6.5pg/mL to about 21.0 pg/mL, about 7.0 pg/mL to about 21.0 pg/mL, about7.5 pg/mL to about 21.0 pg/mL, about 8.0 pg/mL to about 21.0 pg/mL,about 8.5 pg/mL to about 21.0 pg/mL, about 9.0 pg/mL to about 21.0pg/mL, about 9.5 pg/mL to about 21.0 pg/mL, about 10.0 pg/mL to about21.0 pg/mL, about 0.0100 pg/mL to about 20.0 pg/mL, about 0.0200 pg/mLto about 20.0 pg/mL, about 0.0300 pg/mL to about 20.0 pg/mL, about 0.036pg/mL to about 20.0 pg/mL, about 0.0400 pg/mL to about 20.0 pg/mL, about0.0500 pg/mL to about 20.0 pg/mL, about 0.1 pg/mL to about 20.0 pg/mL,about 0.5 pg/mL to about 20.0 pg/mL, about 1.0 pg/mL to about 20.0pg/mL, about 1.5 pg/mL to about 20.0 pg/mL, about 2.0 pg/mL to about20.0 pg/mL, about 2.5 pg/mL to about 20.0 pg/mL, about 3.0 pg/mL toabout 20.0 pg/mL, about 3.5 pg/mL to about 20.0 pg/mL, about 4.0 pg/mLto about 20.0 pg/mL, about 4.5 pg/mL to about 20.0 pg/mL, about 5.0pg/mL to about 20.0 pg/mL, about 5.5 pg/mL to about 20.0 pg/mL, about6.0 pg/mL to about 20.0 pg/mL, about 6.5 pg/mL to about 20.0 pg/mL,about 7.0 pg/mL to about 20.0 pg/mL, about 7.5 pg/mL to about 20.0pg/mL, about 8.0 pg/mL to about 20.0 pg/mL, about 8.5 pg/mL to about20.0 pg/mL, about 9.0 pg/mL to about 20.0 pg/mL, about 9.5 pg/mL toabout 20.0 pg/mL, about 10.0 pg/mL to about 20.0 pg/mL, about 0.0100pg/mL to about 19.0 pg/mL, about 0.0200 pg/mL to about 19.0 pg/mL, about0.0300 pg/mL to about 19.0 pg/mL, about 0.036 pg/mL to about 19.0 pg/mL,about 0.0400 pg/mL to about 19.0 pg/mL, about 0.0500 pg/mL to about 19.0pg/mL, about 0.1 pg/mL to about 19.0 pg/mL, about 0.5 pg/mL to about19.0 pg/mL, about 1.0 pg/mL to about 19.0 pg/mL, about 1.5 pg/mL toabout 19.0 pg/mL, about 2.0 pg/mL to about 19.0 pg/mL, about 2.5 pg/mLto about 19.0 pg/mL, about 3.0 pg/mL to about 19.0 pg/mL, about 3.5pg/mL to about 19.0 pg/mL, about 4.0 pg/mL to about 19.0 pg/mL, about4.5 pg/mL to about 19.0 pg/mL, about 5.0 pg/mL to about 19.0 pg/mL,about 5.5 pg/mL to about 19.0 pg/mL, about 6.0 pg/mL to about 19.0pg/mL, about 6.5 pg/mL to about 19.0 pg/mL, about 7.0 pg/mL to about19.0 pg/mL, about 7.5 pg/mL to about 19.0 pg/mL, about 8.0 pg/mL toabout 19.0 pg/mL, about 8.5 pg/mL to about 19.0 pg/mL, about 9.0 pg/mLto about 19.0 pg/mL, about 9.5 pg/mL to about 19.0 pg/mL, about 10.0pg/mL to about 19.0 pg/mL, about 0.0100 pg/mL to about 18.0 pg/mL, about0.0200 pg/mL to about 18.0 pg/mL, about 0.0300 pg/mL to about 18.0pg/mL, about 0.036 pg/mL to about 18.0 pg/mL, about 0.0400 pg/mL toabout 18.0 pg/mL, about 0.0500 pg/mL to about 18.0 pg/mL, about 0.1pg/mL to about 18.0 pg/mL, about 0.5 pg/mL to about 18.0 pg/mL, about1.0 pg/mL to about 18.0 pg/mL, about 1.5 pg/mL to about 18.0 pg/mL,about 2.0 pg/mL to about 18.0 pg/mL, about 2.5 pg/mL to about 18.0pg/mL, about 3.0 pg/mL to about 18.0 pg/mL, about 3.5 pg/mL to about18.0 pg/mL, about 4.0 pg/mL to about 18.0 pg/mL, about 4.5 pg/mL toabout 18.0 pg/mL, about 5.0 pg/mL to about 18.0 pg/mL, about 5.5 pg/mLto about 18.0 pg/mL, about 6.0 pg/mL to about 18.0 pg/mL, about 6.5pg/mL to about 18.0 pg/mL, about 7.0 pg/mL to about 18.0 pg/mL, about7.5 pg/mL to about 18.0 pg/mL, about 8.0 pg/mL to about 18.0 pg/mL,about 8.5 pg/mL to about 18.0 pg/mL, about 9.0 pg/mL to about 18.0pg/mL, about 9.5 pg/mL to about 18.0 pg/mL, about 10.0 pg/mL to about18.0 pg/mL, about 0.0100 pg/mL to about 17.0 pg/mL, about 0.0200 pg/mLto about 17.0 pg/mL, about 0.0300 pg/mL to about 17.0 pg/mL, about 0.036pg/mL to about 17.0 pg/mL, about 0.0400 pg/mL to about 17.0 pg/mL, about0.0500 pg/mL to about 17.0 pg/mL, about 0.1 pg/mL to about 17.0 pg/mL,about 0.5 pg/mL to about 17.0 pg/mL, about 1.0 pg/mL to about 17.0pg/mL, about 1.5 pg/mL to about 17.0 pg/mL, about 2.0 pg/mL to about17.0 pg/mL, about 2.5 pg/mL to about 17.0 pg/mL, about 3.0 pg/mL toabout 17.0 pg/mL, about 3.5 pg/mL to about 17.0 pg/mL, about 4.0 pg/mLto about 17.0 pg/mL, about 4.5 pg/mL to about 17.0 pg/mL, about 5.0pg/mL to about 17.0 pg/mL, about 5.5 pg/mL to about 17.0 pg/mL, about6.0 pg/mL to about 17.0 pg/mL, about 6.5 pg/mL to about 17.0 pg/mL,about 7.0 pg/mL to about 17.0 pg/mL, about 7.5 pg/mL to about 17.0pg/mL, about 8.0 pg/mL to about 17.0 pg/mL, about 8.5 pg/mL to about17.0 pg/mL, about 9.0 pg/mL to about 17.0 pg/mL, about 9.5 pg/mL toabout 17.0 pg/mL, about 10.0 pg/mL to about 17.0 pg/mL, about 0.0100pg/mL to about 16.0 pg/mL, about 0.0200 pg/mL to about 16.0 pg/mL, about0.0300 pg/mL to about 16.0 pg/mL, about 0.036 pg/mL to about 16.0 pg/mL,about 0.0400 pg/mL to about 16.0 pg/mL, about 0.0500 pg/mL to about 16.0pg/mL, about 0.1 pg/mL to about 16.0 pg/mL, about 0.5 pg/mL to about16.0 pg/mL, about 1.0 pg/mL to about 16.0 pg/mL, about 1.5 pg/mL toabout 16.0 pg/mL, about 2.0 pg/mL to about 16.0 pg/mL, about 2.5 pg/mLto about 16.0 pg/mL, about 3.0 pg/mL to about 16.0 pg/mL, about 3.5pg/mL to about 16.0 pg/mL, about 4.0 pg/mL to about 16.0 pg/mL, about4.5 pg/mL to about 16.0 pg/mL, about 5.0 pg/mL to about 16.0 pg/mL,about 5.5 pg/mL to about 16.0 pg/mL, about 6.0 pg/mL to about 16.0pg/mL, about 6.5 pg/mL to about 16.0 pg/mL, about 7.0 pg/mL to about16.0 pg/mL, about 7.5 pg/mL to about 16.0 pg/mL, about 8.0 pg/mL toabout 16.0 pg/mL, about 8.5 pg/mL to about 16.0 pg/mL, about 9.0 pg/mLto about 16.0 pg/mL, about 9.5 pg/mL to about 16.0 pg/mL, about 10.0pg/mL to about 16.0 pg/mL, about 0.0100 pg/mL to about 15.0 pg/mL, about0.0200 pg/mL to about 15.0 pg/mL, about 0.0300 pg/mL to about 15.0pg/mL, about 0.036 pg/mL to about 15.0 pg/mL, about 0.0400 pg/mL toabout 15.0 pg/mL, about 0.0500 pg/mL to about 15.0 pg/mL, about 0.1pg/mL to about 15.0 pg/mL, about 0.5 pg/mL to about 15.0 pg/mL, about1.0 pg/mL to about 15.0 pg/mL, about 1.5 pg/mL to about 15.0 pg/mL,about 2.0 pg/mL to about 15.0 pg/mL, about 2.5 pg/mL to about 15.0pg/mL, about 3.0 pg/mL to about 15.0 pg/mL, about 3.5 pg/mL to about15.0 pg/mL, about 4.0 pg/mL to about 15.0 pg/mL, about 4.5 pg/mL toabout 15.0 pg/mL, about 5.0 pg/mL to about 15.0 pg/mL, about 5.5 pg/mLto about 15.0 pg/mL, about 6.0 pg/mL to about 15.0 pg/mL, about 6.5pg/mL to about 15.0 pg/mL, about 7.0 pg/mL to about 15.0 pg/mL, about7.5 pg/mL to about 15.0 pg/mL, about 8.0 pg/mL to about 15.0 pg/mL,about 8.5 pg/mL to about 15.0 pg/mL, about 9.0 pg/mL to about 15.0pg/mL, about 9.5 pg/mL to about 15.0 pg/mL or about 10.0 pg/mL to about15.0 pg/mL. In some embodiments, the reference level of cardiac troponinI is about 1.0 pg/mL, about 1.5 pg/mL, about 2.0 pg/mL, about 2.5 pg/mL,about 3.0 pg/mL, about 3.5 pg/mL, about 4.0 pg/mL, about 4.5 pg/mL,about 5.0 pg/mL, about 5.5 pg/mL, about 5.6 pg/mL, about 5.7 pg/mL,about 5.8 pg/mL, about 5.9 pg/mL, about 6.0 pg/mL, about 6.5 pg/mL,about 7.0 pg/mL, about 7.5 pg/mL, about 8.0 pg/mL, about 8.5 pg/mL,about 9.0 pg/mL, about 9.5 pg/mL, or about 10.0 pg/mL.

In some embodiments, the reference age is less than 70 years of age,such as between at least about 35 to about 70 years of age. In someembodiments, the reference age is at least 35 years of age, at least 40years of age, at least 41 years of age, at least 42 years of age, atleast 43 years of age, at least 44 years of age, at least 45 years ofage, at least 46 years of age, at least 47 years of age, at least 48years of age, at least 49 years of age, at least 50 years of age, atleast 55 years of age, at least 60 years of age, at least 65 years ofage, or at least 70 years of age.

In some embodiments, the method further includes treating the humansubject predicted as having an unfavorable outcome with a traumaticbrain injury treatment, as described below. In some embodiments, themethod further includes monitoring the human subject predicted as havingan unfavorable outcome, as described below. In some embodiments, themethod further includes ordering additional tests to obtain furtherclinical information about the mild TBI in the human subject predictedas having an unfavorable outcome. In some embodiments, the methodincludes treating the human subject predicted to have an unfavorableoutcome with a cardioprotective treatment to protect the heart asdescribed below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assaysare described in further detail herein in Sections 11-13. Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. For example, a clinical chemistryformat can include an assay that involves one antibody or no antibody.Examples of analyzers that can be used for the clinical chemistry formatare described in U.S. Patent publication Nos. 2016/0320422 and2015/0112630.

9. Methods of Treating a Human Subject Having Traumatic Brain Injurywith Cardioprotective Therapy

The present disclosure relates, among other methods, to a method fortreating a human subject having or suspected as having a traumatic braininjury. The occurrence of myocardial injury during the acute phase oftraumatic brain injury (TBI) may be a contributor to poor TBI outcome.As such, administering one or more cardioprotective therapies ortherapeutics (e.g., therapies that improve the heart) may improve TBIoutcome and can be employed in the methods described herein. Thesecardioprotective therapies can be administered alone without any othertherapeutics. Alternatively, these cardioprotective therapies can beadministered in combination with other therapeutics administered totreat the TBI, such as those disclosed in Section 10, below.

Specifically, the methods of the disclosure involving one or morecardioprotective therapies or therapeutics includes: a) performing anassay on a sample taken from the human subject within about 24 hoursafter an actual or suspected injury to the head to measure or detect alevel of cardiac troponin I, wherein the sample is a biological sample;and b) providing a cardioprotective therapy or therapeutic to thesubject if the level of cardiac troponin I in the sample is higher thana reference level of cardiac troponin I. In some embodiments, thecardioprotective therapy optionally can include administering one ormore beta-blockers, diuretics, Angiotensin-Converting Enzyme (ACE)inhibitors, calcium channel blockers, lipid lowering therapies, statins(also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors), nitrates, antiplatelets, anticlotting agents,anticoagulation agents, and the like, such as is known in the art. Thenature, amounts and timing for the administration of suchcardioprotective therapies and therapeutics are well known in the art.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of a suspect injury to the head.

Generally, a reference level of cTnI can also be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In some embodiments, the method can include obtaining samples from thesubject and contacting the samples with an antibody for cardiac troponinI to allow formation of a complex of the antibody and cardiac troponinI. The method also includes detecting the resulting antibody-cardiactroponin I complex.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assaysare described in further detail herein in Sections 11-13. Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. For example, a clinical chemistryformat can include an assay that involves one antibody or no antibody.Examples of analyzers that can be used for the clinical chemistry formatare described in U.S. Patent publication Nos. 2016/0320422 and2015/0112630.

10. Treatment and Monitoring of Subjects Suffering from Traumatic BrainInjury

The subject identified or assessed in the methods described above ashaving traumatic brain injury, such as mild traumatic brain injury or amoderate, severe, or moderate to severe traumatic brain injury, may betreated or monitored. In some embodiments, the method further includestreating the human subject assessed as having traumatic brain injurywith a traumatic brain injury treatment, such as any treatments known inthe art. For example, treatment of traumatic brain injury can take avariety of forms depending on the severity of the injury to the head.For example, for subjects suffering from mild TBI, the treatment mayinclude one or more of rest, abstaining from physical activities, suchas sports, avoiding light or wearing sunglasses when out in the light,administration of one or more therapeutics (e.g., such as a medicationfor relief of a headache or migraine, anti-nausea medication, etc).Treatment for patients suffering from a moderate, severe, or moderate tosevere TBI might include administration of one or more appropriatetherapeutics (such as, for example, diuretics, anti-convulsantmedications, medications to sedate and put an individual in adrug-induced coma, or other pharmaceutical or biopharmaceuticalmedications (either known or developed in the future for treatment ofTBI), one or more surgical procedures (such as, for example, removal ofa hematoma, repairing a skull fracture, decompressive craniectomy, etc.)protecting the airway, and one or more therapies (such as, for exampleone or more rehabilitation, physical therapy, occupational therapy,cognitive behavioral therapy, anger management, counseling psychology,etc.). In some embodiments, the method further includes monitoring thehuman subject assessed as having traumatic brain injury (e.g., mild ormoderate, severe, or moderate to severe traumatic). In some embodiments,a subject identified as having traumatic brain injury, such as mildtraumatic brain injury or severe traumatic brain injury, may bemonitored with CT scan or MRI. The treatments employed for mild ormoderate, severe, or moderate to severe TBI described herein can beadministered in connection with one or more cardioprotective therapiesor therapeutics described in Section 9.

11. Methods for Measuring the Level of cTnI

In the methods described above, cTnI levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of cTnI includes contacting thesample with a first specific binding member and second specific bindingmember. In some embodiments the first specific binding member is acapture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of cTnI includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., cTnI-capture antibody), which bindsto an epitope on cTnI or cTnI fragment to form a capture antibody-cTnIantigen complex (e.g., cTnI-capture antibody-cTnI antigen complex), and(2) a detection antibody (e.g., cTnI-detection antibody), which includesa detectable label and binds to an epitope on cTnI that is not bound bythe capture antibody, to form a cTnI antigen-detection antibody complex(e.g., cTnI antigen-cTnI-detection antibody complex), such that acapture antibody-cTnI antigen-detection antibody complex (e.g.,cTnI-capture antibody-cTnI antigen-cTnI-detection antibody complex) isformed, and measuring the amount or concentration of cTnI in the samplebased on the signal generated by the detectable label in the captureantibody-cTnI antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a cTnI antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example the Abbott Laboratoriesinstrument ARCHITECT®, and other core laboratory instruments) other thana point-of-care device may be capable of measuring levels of cTnI in asample at about 0.032 μg/L at 10% CV or lower.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety

12. Cardiac Troponin I Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to cardiac troponin I, such as, for example, humancardiac troponin I (or fragments thereof), referred to as “cardiactroponin I antibody.” Cardiac troponin I antibodies can be used toassess the cardiac troponin I status as a measure of traumatic braininjury, detect the presence of cardiac troponin I in a biologicalsample, quantify the amount of cardiac troponin I present in abiological sample, or detect the presence of and quantify the amount ofcardiac troponin I in a biological sample.

a. Human Cardiac Troponin I (cTnI)

Human cardiac troponin I (cTnI) along with troponin T (TnT) and troponinC (TnC), are the 3 subunits that form the troponin complex of the thinfilaments of striated muscle. Cardiac troponin I is the inhibitorysubunit; blocking actin-myosin interactions and thereby mediatingstriated muscle relaxation. The cTnI subfamily contains three genes:cTnI-skeletal-fast-twitch, cTnI-skeletal-slow-twitch, and cTnI-cardiac.This gene encodes the cTnI-cardiac protein and is exclusively expressedin cardiac muscle tissues.

Human cardiac troponin I may have the following amino acid sequence:

(SEQ ID NO: 1) MADGSSDAAR EPRPAPAPIR RRSSNYRAYA TEPHAKKKSKISASRKLQLK TLLLQIAKQE LEREAEERRG EKGRALSTRCQPLELAGLGF AELQDLCRQL HARVDKVDEE RYDIEAKVTKNITEIADLTQ KIFDLRGKFK RPTLRRVRIS ADAMMQALLGARAKESLDLR AHLKQVKKED TEKENREVGD WRKNIDALSG MEGRKKKFES.

The human cardiac troponin I may be a fragment or variant of SEQ IDNO: 1. The fragment of cardiac troponin I may be between 5 and 210 aminoacids, between 10 and 210 amino acids, between 50 and 210 amino acids,between 60 and 210 amino acids, between 65 and 210 amino acids, between100 and 210 amino acids, between 150 and 210 amino acids, between 100and 210 amino acids, or between 175 and 210 amino acids in length. Thefragment may comprise a contiguous number of amino acids from SEQ ID NO:1.

b. Cardiac Troponin I-Recognizing Antibody

The antibody is an antibody that binds to cardiac troponin I, a fragmentthereof, an epitope of cardiac troponin I, or a variant thereof. Theantibody may be a fragment of the anti-cardiac troponin I antibody or avariant or a derivative thereof. The antibody may be a polyclonal ormonoclonal antibody. The antibody may be a chimeric antibody, a singlechain antibody, an affinity matured antibody, a human antibody, ahumanized antibody, a fully human antibody or an antibody fragment, suchas a Fab fragment, or a mixture thereof. Antibody fragments orderivatives may comprise F(ab′)2, Fv or scFv fragments. The antibodyderivatives can be produced by peptidomimetics. Further, techniquesdescribed for the production of single chain antibodies can be adaptedto produce single chain antibodies.

The anti-cardiac troponin I antibodies may be a chimeric anti-cardiactroponin I or humanized anti-cardiac troponin I antibody. In oneembodiment, both the humanized antibody and chimeric antibody aremonovalent. In one embodiment, both the humanized antibody and chimericantibody comprise a single Fab region linked to an Fc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-cardiac troponin I antibodies. Forexample, libraries of naïve human single chain variable fragments (scFv)may be used to select human anti-cardiac troponin I antibodies.Transgenic animals may be used to express human antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

(1) Epitope

The antibody may immunospecifically bind to human cardiac troponin I(SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibodymay immunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

c. Antibody Preparation/Production

Antibodies may be prepared by any of a variety of techniques, includingthose well known to those skilled in the art. In general, antibodies canbe produced by cell culture techniques, including the generation ofmonoclonal antibodies via conventional techniques, or via transfectionof antibody genes, heavy chains, and/or light chains into suitablebacterial or mammalian cell hosts, in order to allow for the productionof antibodies, wherein the antibodies may be recombinant. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies in either prokaryoticor eukaryotic host cells, expression of antibodies in eukaryotic cellsis preferable, and most preferable in mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesinclude Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody. Recombinant DNA technology may also be used to remove some, orall, of the DNA encoding either or both of the light and heavy chainsthat is not necessary for binding to the antigens of interest. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies. In addition, bifunctional antibodies maybe produced in which one heavy and one light chain are an antibody(i.e., binds human troponin I) and the other heavy and light chain arespecific for an antigen other than human cardiac troponin I bycrosslinking an antibody to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the method of synthesizing a recombinant antibodymay be by culturing a host cell in a suitable culture medium until arecombinant antibody is synthesized. The method can further compriseisolating the recombinant antibody from the culture medium.

Methods of preparing monoclonal antibodies involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Such cell lines may be produced from spleen cells obtainedfrom an immunized animal. The animal may be immunized with cardiactroponin I or a fragment and/or variant thereof. The peptide used toimmunize the animal may comprise amino acids encoding human Fc, forexample the fragment crystallizable region or tail region of humanantibody. The spleen cells may then be immortalized by, for example,fusion with a myeloma cell fusion partner. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thatgrowth of hybrid cells, but not myeloma cells. One such technique useshypoxanthine, aminopterin, thymidine (HAT) selection. Another techniqueincludes electrofusion. After a sufficient time, usually about 1 to 2weeks, colonies of hybrids are observed. Single colonies are selectedand their culture supernatants tested for binding activity against thepolypeptide. Hybridomas having high reactivity and specificity may beused.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. Affinity chromatography is an example ofa method that can be used in a process to purify the antibodies.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment, which comprises bothantigen-binding sites.

The Fv fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecules. TheFv fragment may be derived using recombinant techniques. The Fv fragmentincludes a non-covalent VH::VL heterodimer including an antigen-bindingsite that retains much of the antigen recognition and bindingcapabilities of the native antibody molecule.

The antibody, antibody fragment, or derivative may comprise a heavychain and a light chain complementarity determining region (“CDR”) set,respectively interposed between a heavy chain and a light chainframework (“FR”) set which provide support to the CDRs and define thespatial relationship of the CDRs relative to each other. The CDR set maycontain three hypervariable regions of a heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g.,as available from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al.(1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987)J. Immunol.17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.).; Gray etal. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994)Molec. Biol.Reports 19:125-134 (1994)).

An affinity matured antibody may be produced by any one of a number ofprocedures that are known in the art. For example, see Marks et al.,BioTechnology, 10: 779-783 (1992) describes affinity maturation by VHand VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

Antibody variants can also be prepared using delivering a polynucleotideencoding an antibody to a suitable host such as to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. These methods are known inthe art and are described for example in U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody variants also can be prepared by delivering a polynucleotide toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol.Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies can also beproduced using transgenic plants, according to known methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally, part or all of the non-humanor human CDR sequences are maintained while the non-human sequences ofthe variable and constant regions are replaced with human or other aminoacids.

Small antibody fragments may be diabodies having two antigen-bindingsites, wherein fragments comprise a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (VH VL). See for example, EP 404,097; WO 93/11161; and Hollingeret al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. See also,U.S. Pat. No. 6,632,926 to Chen et al. which is hereby incorporated byreference in its entirety and discloses antibody variants that have oneor more amino acids inserted into a hypervariable region of the parentantibody and a binding affinity for a target antigen which is at leastabout two fold stronger than the binding affinity of the parent antibodyfor the antigen.

The antibody may be a linear antibody. The procedure for making a linearantibody is known in the art and described in Zapata et al., (1995)Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pairof tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The antibodies may be recovered and purified from recombinant cellcultures by known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

It may be useful to detectably label the antibody. Methods forconjugating antibodies to these agents are known in the art. For thepurpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.They can be linked to a cytokine, to a ligand, to another antibody.Suitable agents for coupling to antibodies to achieve an anti-tumoreffect include cytokines, such as interleukin 2 (IL-2) and TumorNecrosis Factor (TNF); photosensitizers, for use in photodynamictherapy, including aluminum (III) phthalocyanine tetrasulfonate,hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131(131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re);antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinesecobra (naja naja atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestrictocin (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; ly207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

Antibody production via the use of hybridoma technology, the selectedlymphocyte antibody method (SLAM), transgenic animals, and recombinantantibody libraries is described in more detail below.

(1) Anti-Cardiac Troponin I Monoclonal Antibodies Using HybridomaTechnology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is alsonoted that the term “monoclonal antibody” as used herein is not limitedto antibodies produced through hybridoma technology. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

Methods of generating monoclonal antibodies as well as antibodiesproduced by the method may comprise culturing a hybridoma cell secretingan antibody of the disclosure wherein, preferably, the hybridoma isgenerated by fusing splenocytes isolated from an animal, e.g., a rat ora mouse, immunized with cardiac troponin I with myeloma cells and thenscreening the hybridomas resulting from the fusion for hybridoma clonesthat secrete an antibody able to bind a polypeptide of the disclosure.Briefly, rats can be immunized with a cardiac troponin I antigen. In apreferred embodiment, the cardiac troponin I antigen is administeredwith an adjuvant to stimulate the immune response. Such adjuvantsinclude complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulating complexes). Such adjuvants mayprotect the polypeptide from rapid dispersal by sequestering it in alocal deposit, or they may contain substances that stimulate the host tosecrete factors that are chemotactic for macrophages and othercomponents of the immune system. Preferably, if a polypeptide is beingadministered, the immunization schedule will involve two or moreadministrations of the polypeptide, spread out over several weeks;however, a single administration of the polypeptide may also be used.

After immunization of an animal with a cardiac troponin I antigen,antibodies and/or antibody-producing cells may be obtained from theanimal. An anti-cardiac troponin I antibody-containing serum is obtainedfrom the animal by bleeding or sacrificing the animal. The serum may beused as it is obtained from the animal, an immunoglobulin fraction maybe obtained from the serum, or the anti-cardiac troponin I antibodiesmay be purified from the serum. Serum or immunoglobulins obtained inthis manner are polyclonal, thus having a heterogeneous array ofproperties.

Once an immune response is detected, e.g., antibodies specific for theantigen cardiac troponin I are detected in the rat serum, the rat spleenis harvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example, cellsfrom cell line SP20 available from the American Type Culture Collection(ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding troponin I.Ascites fluid, which generally contains high levels of antibodies, canbe generated by immunizing rats with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using troponin I, or a portionthereof, or a cell expressing troponin I. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunosorbentassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. Anexample of ELISA screening is provided in PCT Publication No. WO00/37504.

Anti-cardiac troponin I antibody-producing hybridomas are selected,cloned, and further screened for desirable characteristics, includingrobust hybridoma growth, high antibody production, and desirableantibody characteristics. Hybridomas may be cultured and expanded invivo in syngeneic animals, in animals that lack an immune system, e.g.,nude mice, or in cell culture in vitro. Methods of selecting, cloningand expanding hybridomas are well known to those of ordinary skill inthe art.

In a preferred embodiment, hybridomas are rat hybridomas. In anotherembodiment, hybridomas are produced in a non-human, non-rat species suchas mice, sheep, pigs, goats, cattle, or horses. In yet another preferredembodiment, the hybridomas are human hybridomas, in which a humannon-secretory myeloma is fused with a human cell expressing ananti-cardiac troponin I antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of thedisclosure may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce two identical Fabfragments) or pepsin (to produce an F(ab′)2 fragment). A F(ab′)2fragment of an IgG molecule retains the two antigen-binding sites of thelarger (“parent”) IgG molecule, including both light chains (containingthe variable light chain and constant light chain regions), the CH1domains of the heavy chains, and a disulfide-forming hinge region of theparent IgG molecule. Accordingly, an F(ab′)2 fragment is still capableof crosslinking antigen molecules like the parent IgG molecule.

(2) Anti-Cardiac Troponin I Monoclonal Antibodies Using SLAM

In another aspect of the disclosure, recombinant antibodies aregenerated from single, isolated lymphocytes using a procedure referredto in the art as the selected lymphocyte antibody method (SLAM), asdescribed in U.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551;and Babcook et al., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). Inthis method, single cells secreting antibodies of interest, e.g.,lymphocytes derived from any one of the immunized animals are screenedusing an antigen-specific hemolytic plaque assay, wherein the antigencardiac troponin I, a subunit of cardiac troponin I, or a fragmentthereof, is coupled to sheep red blood cells using a linker, such asbiotin, and used to identify single cells that secrete antibodies withspecificity for cardiac troponin I. Following identification ofantibody-secreting cells of interest, heavy- and light-chain variableregion cDNAs are rescued from the cells by reverse transcriptase-PCR(RT-PCR) and these variable regions can then be expressed, in thecontext of appropriate immunoglobulin constant regions (e.g., humanconstant regions), in mammalian host cells, such as COS or CHO cells.The host cells transfected with the amplified immunoglobulin sequences,derived from in vivo selected lymphocytes, can then undergo furtheranalysis and selection in vitro, for example, by panning the transfectedcells to isolate cells expressing antibodies to cardiac troponin I. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation method. See, for example, PCTPublication No. WO 97/29131 and PCT Publication No. WO 00/56772.

(3) Anti-Cardiac Troponin I Monoclonal Antibodies Using TransgenicAnimals

In another embodiment of the disclosure, antibodies are produced byimmunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with a cardiac troponin I antigen. In anembodiment, the non-human animal is a transgenic mouse (XENOMOUSE®), anengineered mouse strain that comprises large fragments of the humanimmunoglobulin loci and is deficient in mouse antibody production. See,e.g., Green et al., Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos.5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001;6,114,598; and 6,130,364. See also PCT Publication Nos. WO 91/10741; WO94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. TheXENOMOUSE® transgenic mouse produces an adult-like human repertoire offully human antibodies, and generates antigen-specific human monoclonalantibodies. The XENOMOUSE® transgenic mouse contains approximately 80%of the human antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci and xlight chain loci. See Mendez et al., Nature Genetics, 15: 146-156(1997), Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), thedisclosures of which are hereby incorporated by reference.

(4) Anti-Cardiac Troponin I Monoclonal Antibodies Using RecombinantAntibody Libraries

In vitro methods also can be used to make the antibodies of thedisclosure, wherein an antibody library is screened to identify anantibody having the desired cardiac troponin I-binding specificity.Methods for such screening of recombinant antibody libraries are wellknown in the art and include methods described in, for example, U.S.Pat. No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92/18619(Kang et al.); PCT Publication No. WO 91/17271 (Dower et al.); PCTPublication No. WO 92/20791 (Winter et al.); PCT Publication No. WO92/15679 (Markland et al.); PCT Publication No. WO 93/01288 (Breitlinget al.); PCT Publication No. WO 92/01047 (McCafferty et al.); PCTPublication No. WO 92/09690 (Garrard et al.); Fuchs et al.,Bio/Technology, 9: 1369-1372 (1991); Hay et al., Hum. Antibod.Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281(1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths etal., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991); Gram etal., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992); Garrard et al.,Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nucl. AcidsRes., 19: 4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991); U.S. Patent Application Publication No.2003/0186374; and PCT Publication No. WO 97/29131, the contents of eachof which are incorporated herein by reference.

The recombinant antibody library may be from a subject immunized withcardiac troponin I, or a portion of cardiac troponin I. Alternatively,the recombinant antibody library may be from a naive subject, i.e., onewho has not been immunized with cardiac troponin I, such as a humanantibody library from a human subject who has not been immunized withhuman cardiac troponin I. Antibodies of the disclosure are selected byscreening the recombinant antibody library with the peptide comprisinghuman cardiac troponin I to thereby select those antibodies thatrecognize troponin I. Methods for conducting such screening andselection are well known in the art, such as described in the referencesin the preceding paragraph. To select antibodies of the disclosurehaving particular binding affinities for cardiac troponin I, such asthose that dissociate from human cardiac troponin I with a particularK_(off) rate constant, the art-known method of surface plasmon resonancecan be used to select antibodies having the desired K_(off) rateconstant. To select antibodies of the disclosure having a particularneutralizing activity for cardiac troponin I, such as those with aparticular IC₅₀, standard methods known in the art for assessing theinhibition of cardiac troponin I activity may be used.

In one aspect, the disclosure pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human cardiac troponin I.Preferably, the antibody is a neutralizing antibody. In variousembodiments, the antibody is a recombinant antibody or a monoclonalantibody.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. Such phage can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Phageexpressing an antigen binding domain that binds the antigen of interestcan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv, or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies include those disclosed in Brinkmannet al., J Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al.,Advances in Immunology, 57: 191-280 (1994); PCT Publication No. WO92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743; and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al.,Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al.,Science, 240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the disclosure. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98/31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology is PROfusion display technology.

In another approach, the antibodies can also be generated using yeastdisplay methods known in the art. In yeast display methods, geneticmethods are used to tether antibody domains to the yeast cell wall anddisplay them on the surface of yeast. In particular, such yeast can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Examples ofyeast display methods that can be used to make the antibodies includethose disclosed in U.S. Pat. No. 6,699,658 (Wittrup et al.) incorporatedherein by reference.

d. Production of Recombinant Cardiac Troponin I Antibodies

Antibodies may be produced by any of a number of techniques known in theart. For example, expression from host cells, wherein expressionvector(s) encoding the heavy and light chains is (are) transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection, and the like. Although it ispossible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the disclosure include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody of this disclosure. Recombinant DNA technology may also be usedto remove some, or all, of the DNA encoding either or both of the lightand heavy chains that is not necessary for binding to the antigens ofinterest. The molecules expressed from such truncated DNA molecules arealso encompassed by the antibodies of the disclosure. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the disclosure (i.e., binds human cardiactroponin I) and the other heavy and light chain are specific for anantigen other than human cardiac troponin I by crosslinking an antibodyof the disclosure to a second antibody by standard chemical crosslinkingmethods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the disclosure, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the disclosure provides a method of synthesizinga recombinant antibody of the disclosure by culturing a host cell of thedisclosure in a suitable culture medium until a recombinant antibody ofthe disclosure is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

(1) Humanized Antibody

The humanized antibody may be an antibody or a variant, derivative,analog or portion thereof which immunospecifically binds to an antigenof interest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. The humanized antibody may befrom a non-human species antibody that binds the desired antigen havingone or more complementarity determining regions (CDRs) from thenon-human species and framework regions from a human immunoglobulinmolecule.

As used herein, the term “substantially” in the context of a CDR refersto a CDR having an amino acid sequence at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′,F(ab′)₂, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. According to one aspect, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or of a heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3, and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In one embodiment, suchmutations, however, will not be extensive. Usually, at least 90%, atleast 95%, at least 98%, or at least 99% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences.As used herein, the term “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence. As used herein, theterm “consensus immunoglobulin sequence” refers to the sequence formedfrom the most frequently occurring amino acids (or nucleotides) in afamily of related immunoglobulin sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In afamily of immunoglobulins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

The humanized antibody may be designed to minimize unwantedimmunological response toward rodent anti-human antibodies, which limitsthe duration and effectiveness of therapeutic applications of thosemoieties in human recipients. The humanized antibody may have one ormore amino acid residues introduced into it from a source that isnon-human. These non-human residues are often referred to as “import”residues, which are typically taken from a variable domain. Humanizationmay be performed by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. For example, see U.S.Pat. No. 4,816,567, the contents of which are herein incorporated byreference. The humanized antibody may be a human antibody in which somehypervariable region residues, and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.Humanization or engineering of antibodies of the present disclosure canbe performed using any known method, such as but not limited to thosedescribed in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483;5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023;6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

The humanized antibody may retain high affinity for cardiac troponin Iand other favorable biological properties. The humanized antibody may beprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available. Computer programs are available thatillustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristics, such as increased affinity forcardiac troponin I, is achieved. In general, the hypervariable regionresidues may be directly and most substantially involved in influencingantigen binding.

As an alternative to humanization, human antibodies (also referred toherein as “fully human antibodies”) can be generated. For example, it ispossible to isolate human antibodies from libraries via PROfusion and/oryeast related technologies. It is also possible to produce transgenicanimals (e.g., mice that are capable, upon immunization, of producing afull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. The humanized or fully humanantibodies may be prepared according to the methods described in U.S.Pat. Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517;6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;6,682,928; and 6,984,720, the contents each of which are hereinincorporated by reference.

e. Anti-Cardiac Troponin I Antibodies

Anti-cardiac troponin I antibodies may be generated using the techniquesdescribed above as well as using routine techniques known in the art. Insome embodiments, the anti-cardiac troponin I antibody may be anunconjugated cardiac troponin I antibody, such as cardiac troponin Iantibodies available from Abcam (such as Anti-Cardiac troponin Iantibody (ab47003)), Thermofisher (such as cardiac troponin I monoclonalantibody (12F10), cardiac troponin I polyclonal antibody, cardiactroponin I antibody (1HCLC), ABFINITY™ rabbit oligoclonal, cardiactroponin I antibody (1H11L19), ABFINITY™ rabbit monoclonal), Santa Cruz(such as cardiac troponin I antibody (C-4) (Catalog number sc-133117),cardiac troponin I antibody (4) (Catalog number sc-130351), cardiactroponin I antibody (12) (Catalog number sc-130350), cardiac troponin Iantibody (H-170) (Catalog number sc-15368), cardiac troponin I antibody(C-19) (Catalog number sc-8118), cardiac troponin I-C antibody (G-11)(Catalog number sc-376662), cardiac troponin I-C antibody (M46) (Catalognumber sc-52277), cardiac troponin I-C antibody (10B11) (Catalog numbersc-52266) and hytest (Monoclonal mouse anti-cardiac cardiac troponin I(catalog number 4T21).

13. Variations on Methods

The disclosed methods of determining the presence or amount of analyteof interest (e.g., cTnI) present in a sample may be as described herein.The methods may also be adapted in view of other methods for analyzinganalytes. Examples of well-known variations include, but are not limitedto, immunoassay, such as sandwich immunoassay (e.g.,monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonalsandwich immunoassays, including enzyme detection (enzyme immunoassay(EIA) or enzyme-linked immunosorbent assay (ELISA), competitiveinhibition immunoassay (e.g., forward and reverse), enzyme multipliedimmunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc.

a. Immunoassay

The analyte of interest, and/or peptides of fragments thereof (e.g.,cTnI and/or peptides or fragments thereof, i.e., cTnI fragments), may beanalyzed using cTnI antibodies in an immunoassay. The presence or amountof analyte (e.g., cTnI) can be determined using antibodies and detectingspecific binding to the analyte (e.g., cTnI). For example, the antibody,or antibody fragment thereof, may specifically bind to the analyte(e.g., cTnI). If desired, one or more of the antibodies can be used incombination with one or more commercially availablemonoclonal/polyclonal antibodies. Such antibodies are available fromcompanies such as R&D Systems, Inc. (Minneapolis, Minn.) and Enzo LifeSciences International, Inc. (Plymouth Meeting, Pa.).

The presence or amount of analyte (e.g., cTnI) present in a body samplemay be readily determined using an immunoassay, such as sandwichimmunoassay (e.g., monoclonal-monoclonal sandwich immunoassays,monoclonal-polyclonal sandwich immunoassays, including radioisotopedetection (radioimmunoassay (RIA)) and enzyme detection (enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g.,Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)). An exampleof a point-of-care device that can be used is i-STAT® (Abbott,Laboratories, Abbott Park, Ill.). Other methods that can be used includea chemiluminescent microparticle immunoassay, in particular oneemploying the ARCHITECT® automated analyzer (Abbott Laboratories, AbbottPark, Ill.), as an example. Other methods include, for example, massspectrometry, and immunohistochemistry (e.g., with sections from tissuebiopsies), using anti-analyte (e.g., anti-cTnI) antibodies (monoclonal,polyclonal, chimeric, humanized, human, etc.) or antibody fragmentsthereof against analyte (e.g., cTnI). Other methods of detection includethose described in, for example, U.S. Pat. Nos. 6,143,576; 6,113,855;6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527;5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of whichis hereby incorporated by reference in its entirety. Specificimmunological binding of the antibody to the analyte (e.g., cTnI) can bedetected via direct labels, such as fluorescent or luminescent tags,metals and radionuclides attached to the antibody or via indirectlabels, such as alkaline phosphatase or horseradish peroxidase.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, the surface of an assay plate (such as microtiter wells),pieces of a solid substrate material, and the like. An assay strip canbe prepared by coating the antibody or plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

A homogeneous format may be used. For example, after the test sample isobtained from a subject, a mixture is prepared. The mixture contains thetest sample being assessed for analyte (e.g., cTnI) and a specificbinding partner. The order in which the test sample and the specificbinding partner are added to form the mixture is not critical. The testsample is simultaneously contacted with the specific binding partner. Insome embodiments, the specific binding partner and any cTnI contained inthe test sample may form a specific binding partner-analyte (e.g.,cTnI)-antigen complex. The specific binding partner may be ananti-analyte antibody (e.g., anti-cTnI antibody that binds to an epitopehaving an amino acid sequence comprising at least three contiguous (3)amino acids of SEQ ID NO: 1. Moreover, the specific binding partner maybe labeled with or contains a detectable label as described above.

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for analyte (e.g., cTnI) and afirst specific binding partner, wherein the first specific bindingpartner and any cTnI contained in the test sample form a first specificbinding partner-analyte (e.g., cTnI)-antigen complex. The first specificbinding partner may be an anti-analyte antibody (e.g., anti-cTnIantibody that binds to an epitope having an amino acid sequencecomprising at least three contiguous (3) amino acids of SEQ ID NO: 1).The order in which the test sample and the first specific bindingpartner are added to form the mixture is not critical.

The first specific binding partner may be immobilized on a solid phase.The solid phase used in the immunoassay (for the specific bindingpartner) can be any solid phase known in the art, such as, but notlimited to, a magnetic particle, a bead, a test tube, a microtiterplate, a cuvette, a membrane, a scaffolding molecule, a film, a filterpaper, a disc, and a chip. In those embodiments where the solid phase isa bead, the bead may be a magnetic bead or a magnetic particle. Magneticbeads/particles may be ferromagnetic, ferrimagnetic, paramagnetic,superparamagnetic or ferrofluidic. Exemplary ferromagnetic materialsinclude Fe, Co, Ni, Gd, Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examplesof ferrimagnetic materials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (orFeO.Fe₂O₃). Beads can have a solid core portion that is magnetic and issurrounded by one or more non-magnetic layers. Alternately, the magneticportion can be a layer around a non-magnetic core. The solid support onwhich the first specific binding member is immobilized may be stored indry form or in a liquid. The magnetic beads may be subjected to amagnetic field prior to or after contacting with the sample with amagnetic bead on which the first specific binding member is immobilized.

After the mixture containing the first specific binding partner-analyte(e.g., cTnI) antigen complex is formed, any unbound analyte (e.g., cTnI)is removed from the complex using any technique known in the art. Forexample, the unbound analyte (e.g., cTnI) can be removed by washing.Desirably, however, the first specific binding partner is present inexcess of any analyte (e.g., cTnI) present in the test sample, such thatall analyte (e.g., cTnI) that is present in the test sample is bound bythe first specific binding partner.

After any unbound analyte (e.g., cTnI) is removed, a second specificbinding partner is added to the mixture to form a first specific bindingpartner-analyte of interest (e.g., cTnI)-second specific binding partnercomplex. The second specific binding partner may be an anti-analyteantibody (e.g., cTnI antibody that binds to an epitope having an aminoacid sequence comprising at least three contiguous (3) amino acids ofSEQ ID NO: 1). Moreover, the second specific binding partner is labeledwith or contains a detectable label as described above.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles (such as a magnetic bead), latex particles or modified surfacelatex particles, polymer or polymer film, plastic or plastic film,planar substrate, the surface of an assay plate (such as microtiterwells), pieces of a solid substrate material, and the like. An assaystrip can be prepared by coating the antibody or plurality of antibodiesin an array on a solid support. This strip can then be dipped into thetest sample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

(1) Sandwich Immunoassay

A sandwich immunoassay measures the amount of antigen between two layersof antibodies (i.e., at least one capture antibody) and a detectionantibody (i.e., at least one detection antibody). The capture antibodyand the detection antibody bind to different epitopes on the antigen,e.g., analyte of interest such as cTnI). Desirably, binding of thecapture antibody to an epitope does not interfere with binding of thedetection antibody to an epitope. Either monoclonal or polyclonalantibodies may be used as the capture and detection antibodies in thesandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte (e.g., cTnI) in a test sample. More specifically, the at leasttwo antibodies bind to certain epitopes of analyte (e.g., cTnI) formingan immune complex which is referred to as a “sandwich”. One or moreantibodies can be used to capture the analyte (e.g., cTnI) in the testsample (these antibodies are frequently referred to as a “capture”antibody or “capture” antibodies) and one or more antibodies is used tobind a detectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection” antibody or“detection” antibodies). In a sandwich assay, the binding of an antibodyto its epitope desirably is not diminished by the binding of any otherantibody in the assay to its respective epitope. Antibodies are selectedso that the one or more first antibodies brought into contact with atest sample suspected of containing analyte (e.g., cTnI) do not bind toall or part of an epitope recognized by the second or subsequentantibodies, thereby interfering with the ability of the one or moresecond detection antibodies to bind to the analyte (e.g., cTnI).

The antibodies may be used as a first antibody in said immunoassay. Theantibody immunospecifically binds to epitopes on analyte (e.g., cTnI).In addition to the antibodies of the present disclosure, saidimmunoassay may comprise a second antibody that immunospecifically bindsto epitopes that are not recognized or bound by the first antibody.

A test sample suspected of containing analyte (e.g., cTnI) can becontacted with at least one first capture antibody (or antibodies) andat least one second detection antibodies either simultaneously orsequentially. In the sandwich assay format, a test sample suspected ofcontaining analyte (e.g., cTnI) is first brought into contact with theat least one first capture antibody that specifically binds to aparticular epitope under conditions which allow the formation of a firstantibody-analyte (e.g., cTnI) antigen complex. If more than one captureantibody is used, a first multiple capture antibody-cTnI antigen complexis formed. In a sandwich assay, the antibodies, preferably, the at leastone capture antibody, are used in molar excess amounts of the maximumamount of analyte (e.g., cTnI) expected in the test sample. For example,from about 5 μg/mL to about 1 mg/mL of antibody per ml of microparticlecoating buffer may be used.

i. Anti-cTnI Capture Antibody

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one first capture antibody can bebound to a solid support which facilitates the separation the firstantibody-analyte (e.g., cTnI) complex from the test sample. Any solidsupport known in the art can be used, including but not limited to,solid supports made out of polymeric materials in the forms of wells,tubes, or beads (such as a microparticle). The antibody (or antibodies)can be bound to the solid support by adsorption, by covalent bondingusing a chemical coupling agent or by other means known in the art,provided that such binding does not interfere with the ability of theantibody to bind analyte (e.g., cTnI). Moreover, if necessary, the solidsupport can be derivatized to allow reactivity with various functionalgroups on the antibody. Such derivatization requires the use of certaincoupling agents such as, but not limited to, maleic anhydride,N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte (e.g., cTnI) isincubated in order to allow for the formation of a first captureantibody (or multiple antibody)-analyte (e.g., cTnI) complex. Theincubation can be carried out at a pH of from about 4.5 to about 10.0,at a temperature of from about 2° C. to about 45° C., and for a periodfrom at least about one (1) minute to about eighteen (18) hours, fromabout 2-6 minutes, from about 7-12 minutes, from about 5-15 minutes, orfrom about 3-4 minutes.

ii. Detection Antibody

After formation of the first/multiple capture antibody-analyte (e.g.,cTnI) complex, the complex is then contacted with at least one seconddetection antibody (under conditions that allow for the formation of afirst/multiple antibody-analyte (e.g., cTnI) antigen-second antibodycomplex). In some embodiments, the test sample is contacted with thedetection antibody simultaneously with the capture antibody. If thefirst antibody-analyte (e.g., cTnI) complex is contacted with more thanone detection antibody, then a first/multiple capture antibody-analyte(e.g., cTnI)-multiple antibody detection complex is formed. As withfirst antibody, when the at least second (and subsequent) antibody isbrought into contact with the first antibody-analyte (e.g., cTnI)complex, a period of incubation under conditions similar to thosedescribed above is required for the formation of the first/multipleantibody-analyte (e.g., cTnI)-second/multiple antibody complex.Preferably, at least one second antibody contains a detectable label.The detectable label can be bound to the at least one second antibodyprior to, simultaneously with or after the formation of thefirst/multiple antibody-analyte (e.g., cTnI)-second/multiple antibodycomplex. Any detectable label known in the art can be used.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith an acridinium compound, detectably labeled first specific bindingpartner-antigen (e.g., cTnI) complexes form. Alternatively, if a secondspecific binding partner is used and the second specific binding partneris detectably labeled with an acridinium compound, detectably labeledfirst specific binding partner-analyte (e.g., cTnI)-second specificbinding partner complexes form. Any unbound specific binding partner,whether labeled or unlabeled, can be removed from the mixture using anytechnique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture before, simultaneously with, or after theaddition of an above-described acridinium compound. Hydrogen peroxidecan be generated in situ in a number of ways such as would be apparentto one skilled in the art.

Alternatively, a source of hydrogen peroxide can be simply added to themixture. For example, the source of the hydrogen peroxide can be one ormore buffers or other solutions that are known to contain hydrogenperoxide. In this regard, a solution of hydrogen peroxide can simply beadded.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte (e.g., cTnI) is generated.The basic solution contains at least one base and has a pH greater thanor equal to 10, preferably, greater than or equal to 12. Examples ofbasic solutions include, but are not limited to, sodium hydroxide,potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesiumhydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide,calcium carbonate, and calcium bicarbonate. The amount of basic solutionadded to the sample depends on the concentration of the basic solution.Based on the concentration of the basic solution used, one skilled inthe art can easily determine the amount of basic solution to add to thesample. Other labels other than chemiluminescent labels can be employed.For instance, enzymatic labels (including but not limited to alkalinephosphatase) can be employed.

The chemiluminescent signal, or other signal, that is generated can bedetected using routine techniques known to those skilled in the art.Based on the intensity of the signal generated, the amount of analyte ofinterest (e.g., cTnI) in the sample can be quantified. Specifically, theamount of analyte (e.g., cTnI) in the sample is proportional to theintensity of the signal generated. The amount of analyte (e.g., cTnI)present can be quantified by comparing the amount of light generated toa standard curve for analyte (e.g., cTnI) or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions of known concentrations of analyte (e.g., cTnI)by mass spectroscopy, gravimetric methods, and other techniques known inthe art.

(2) Forward Competitive Inhibition Assay

In a forward competitive format, an aliquot of labeled analyte ofinterest (e.g., cTnI) having a fluorescent label, a tag attached with acleavable linker, etc.) of a known concentration is used to compete withanalyte of interest (e.g., cTnI) in a test sample for binding to analyteof interest antibody (e.g., a cTnI antibody).

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and a labeled analyte of interest,analyte of interest fragment or analyte of interest variant thereof. Theanalyte of interest peptide, analyte of interest fragment or analyte ofinterest variant can be labeled with any detectable label, including adetectable label comprised of tag attached with a cleavable linker. Inthis assay, the antibody can be immobilized on to a solid support.Alternatively, the antibody can be coupled to an antibody, such as anantispecies antibody, that has been immobilized on a solid support, suchas a microparticle or planar substrate.

The labeled analyte of interest, the test sample and the antibody areincubated under conditions similar to those described above inconnection with the sandwich assay format. Two different species ofantibody-analyte of interest complexes may then be generated.Specifically, one of the antibody-analyte of interest complexesgenerated contains a detectable label (e.g., a fluorescent label, etc.)while the other antibody-analyte of interest complex does not contain adetectable label. The antibody-analyte of interest complex can be, butdoes not have to be, separated from the remainder of the test sampleprior to quantification of the detectable label. Regardless of whetherthe antibody-analyte of interest complex is separated from the remainderof the test sample, the amount of detectable label in theantibody-analyte of interest complex is then quantified. Theconcentration of analyte of interest (such as membrane-associatedanalyte of interest, soluble analyte of interest, fragments of solubleanalyte of interest, variants of analyte of interest(membrane-associated or soluble analyte of interest) or any combinationsthereof) in the test sample can then be determined, e.g., as describedabove.

(3) Reverse Competitive Inhibition Assay

In a reverse competition assay, an immobilized analyte of interest(e.g., cTnI) can either be sequentially or simultaneously contacted witha test sample and at least one labeled antibody.

The analyte of interest can be bound to a solid support, such as thesolid supports discussed above in connection with the sandwich assayformat.

The immobilized analyte of interest, test sample and at least onelabeled antibody are incubated under conditions similar to thosedescribed above in connection with the sandwich assay format. Twodifferent species analyte of interest-antibody complexes are thengenerated. Specifically, one of the analyte of interest-antibodycomplexes generated is immobilized and contains a detectable label(e.g., a fluorescent label, etc.) while the other analyte ofinterest-antibody complex is not immobilized and contains a detectablelabel. The non-immobilized analyte of interest-antibody complex and theremainder of the test sample are removed from the presence of theimmobilized analyte of interest-antibody complex through techniquesknown in the art, such as washing. Once the non-immobilized analyte ofinterest antibody complex is removed, the amount of detectable label inthe immobilized analyte of interest-antibody complex is then quantifiedfollowing cleavage of the tag. The concentration of analyte of interestin the test sample can then be determined by comparing the quantity ofdetectable label as described above.

(4) One-Step Immunoassay or “Capture on the Fly” Assay

In a capture on the fly immunoassay, a solid substrate is pre-coatedwith an immobilization agent. The capture agent, the analyte (e.g.,cTnI) and the detection agent are added to the solid substrate together,followed by a wash step prior to detection. The capture agent can bindthe analyte (e.g., cTnI) and comprises a ligand for an immobilizationagent. The capture agent and the detection agents may be antibodies orany other moiety capable of capture or detection as described herein orknown in the art. The ligand may comprise a peptide tag and animmobilization agent may comprise an anti-peptide tag antibody.Alternately, the ligand and the immobilization agent may be any pair ofagents capable of binding together so as to be employed for a capture onthe fly assay (e.g., specific binding pair, and others such as are knownin the art). More than one analyte may be measured. In some embodiments,the solid substrate may be coated with an antigen and the analyte to beanalyzed is an antibody.

In certain other embodiments, in a one-step immunoassay or “capture onthe fly”, a solid support (such as a microparticle) pre-coated with animmobilization agent (such as biotin, streptavidin, etc.) and at least afirst specific binding member and a second specific binding member(which function as capture and detection reagents, respectively) areused. The first specific binding member comprises a ligand for theimmobilization agent (for example, if the immobilization agent on thesolid support is streptavidin, the ligand on the first specific bindingmember may be biotin) and also binds to the analyte of interest (e.g.,cTnI). The second specific binding member comprises a detectable labeland binds to an analyte of interest (e.g., cTnI). The solid support andthe first and second specific binding members may be added to a testsample (either sequentially or simultaneously). The ligand on the firstspecific binding member binds to the immobilization agent on the solidsupport to form a solid support/first specific binding member complex.Any analyte of interest present in the sample binds to the solidsupport/first specific binding member complex to form a solidsupport/first specific binding member/analyte complex. The secondspecific binding member binds to the solid support/first specificbinding member/analyte complex and the detectable label is detected. Anoptional wash step may be employed before the detection. In certainembodiments, in a one-step assay more than one analyte may be measured.In certain other embodiments, more than two specific binding members canbe employed. In certain other embodiments, multiple detectable labelscan be added. In certain other embodiments, multiple analytes ofinterest can be detected, or their amounts, levels or concentrations,measured, determined or assessed.

The use of a capture on the fly assay can be done in a variety offormats as described herein, and known in the art. For example theformat can be a sandwich assay such as described above, but alternatelycan be a competition assay, can employ a single specific binding member,or use other variations such as are known.

14. Other Factors

The methods of diagnosing, prognosticating, and/or assessing, asdescribed above, can further include using other factors for thediagnosis, prognostication, and assessment. In some embodiments,traumatic brain injury may be diagnosed using the Glasgow Coma Scale orthe Extended Glasgow Outcome Scale (GOSE). Other tests, scales orindices can also be used either alone or in combination with the GlasgowComa Scale. An example is the Ranchos Los Amigos Scale. The Ranchos LosAmigos Scale measures the levels of awareness, cognition, behavior andinteraction with the environment. The Ranchos Los Amigos Scale includes:Level I: No Response; Level II: Generalized Response; Level III:Localized Response; Level IV: Confused-agitated; Level V:Confused-inappropriate; Level VI: Confused-appropriate; Level VII:Automatic-appropriate; and Level VIII: Purposeful-appropriate.

15. Samples

In some embodiments, the sample is obtained after the human subjectsustained an injury to the head caused by physical shaking, blunt impactby an external mechanical or other force that results in a closed oropen head trauma, one or more falls, explosions or blasts or other typesof blunt force trauma. In some embodiments, the sample is obtained afterthe human subject has ingested or been exposed to a chemical, toxin orcombination of a chemical and toxin. Examples of such chemicals and/ortoxins include, fires, molds, asbestos, pesticides and insecticides,organic solvents, paints, glues, gases (such as carbon monoxide,hydrogen sulfide, and cyanide), organic metals (such as methyl mercury,tetraethyl lead and organic tin) and/or one or more drugs of abuse. Insome embodiments, the sample is obtained from a human subject thatsuffers from an autoimmune disease, a metabolic disorder, a brain tumor,hypoxia, one or more viruses, meningitis, hydrocephalus or combinationsthereof.

In yet another embodiment, the methods described herein use samples thatalso can be used to determine whether or not a subject has or is at riskof developing mild traumatic brain injury by determining the levels ofcTnI in a subject using the anti-cTnI antibodies described below, orantibody fragments thereof. Thus, in particular embodiments, thedisclosure also provides a method for determining whether a subjecthaving, or at risk for, traumatic brain injuries, discussed herein andknown in the art, is a candidate for therapy or treatment. Generally,the subject is at least one who: (i) has experienced an injury to thehead; (ii) ingested and/or been exposed to one or more chemicals and/ortoxins; (iii) suffers from an autoimmune disease, a metabolic disorder,a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalusor suffers from any combinations thereof; or (iv) any combinations of(i)-(iii); or, who has actually been diagnosed as having, or being atrisk for TBI (such as, for example, subjects suffering from anautoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one ormore viruses, meningitis, hydrocephalus or combinations thereof), and/orwho demonstrates an unfavorable (i.e., clinically undesirable)concentration or amount of cTnI or cTnI fragment, as described herein.

a. Test or Biological Sample

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing cTnI. The sample maybe derived from any suitable source. In some cases, the sample maycomprise a liquid, fluent particulate solid, or fluid suspension ofsolid particles. In some cases, the sample may be processed prior to theanalysis described herein. For example, the sample may be separated orpurified from its source prior to analysis; however, in certainembodiments, an unprocessed sample containing cTnI may be assayeddirectly. In a particular example, the source containing cTnI is a humanbodily substance (e.g., bodily fluid, blood such as whole blood, serum,plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid,lymph fluid, amniotic fluid, interstitial fluid, lung lavage,cerebrospinal fluid, feces, tissue, organ, or the like). Tissues mayinclude, but are not limited to skeletal muscle tissue, liver tissue,lung tissue, kidney tissue, myocardial tissue, brain tissue, bonemarrow, cervix tissue, skin, etc. The sample may be a liquid sample or aliquid extract of a solid sample. In certain cases, the source of thesample may be an organ or tissue, such as a biopsy sample, which may besolubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or thelike. In some cases, the volume of the fluid sample is between about0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, betweenabout 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source containing cTnI is a humanbody fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,prior to use. In other cases, the fluid sample is not diluted prior touse in an assay.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source containing cTnI is a human body fluid(e.g., blood, serum), the fluid may be concentrated by precipitation,evaporation, filtration, centrifugation, or a combination thereof. Afluid sample may be concentrated about 1-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about100-fold, or greater, prior to use.

b. Controls

It may be desirable to include a control sample. The control sample maybe analyzed concurrently with the sample from the subject as describedabove. The results obtained from the subject sample can be compared tothe results obtained from the control sample. Standard curves may beprovided, with which assay results for the sample may be compared. Suchstandard curves present levels of marker as a function of assay units,i.e., fluorescent signal intensity, if a fluorescent label is used.Using samples taken from multiple donors, standard curves can beprovided for reference levels of the cTnI in normal healthy tissue, aswell as for “at-risk” levels of the cTnI in tissue taken from donors,who may have one or more of the characteristics set forth above.

Thus, in view of the above, a method for determining the presence,amount, or concentration of cTnI in a test sample is provided. Themethod comprises assaying the test sample for cTnI by an immunoassay,for example, employing at least one capture antibody that binds to anepitope on cTnI and at least one detection antibody that binds to anepitope on cTnI which is different from the epitope for the captureantibody and optionally includes a detectable label, and comprisingcomparing a signal generated by the detectable label as a direct orindirect indication of the presence, amount or concentration of cTnI inthe test sample to a signal generated as a direct or indirect indicationof the presence, amount or concentration of cTnI in a calibrator. Thecalibrator is optionally, and is preferably, part of a series ofcalibrators in which each of the calibrators differs from the othercalibrators in the series by the concentration of cTnI.

16. Kit

Provided herein is a kit, which may be used for assaying or assessing atest sample for cTnI and/or cTnI fragment. The kit comprises at leastone component for assaying the test sample for cTnI and instructions forassaying the test sample for cTnI. For example, the kit can compriseinstructions for assaying the test sample for cTnI by immunoassay, e.g.,chemiluminescent microparticle immunoassay. Instructions included inkits can be affixed to packaging material or can be included as apackage insert. While the instructions are typically written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this disclosure. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

The at least one component may include at least one compositioncomprising one or more isolated antibodies or antibody fragments thereofthat specifically bind to cTnI. The antibody may be a cTnI detectionantibody and/or capture antibody.

Alternatively or additionally, the kit can comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized, cTnI and/or atleast one container (e.g., tube, microtiter plates or strips, which canbe already coated with an anti-cTnI antibody) for conducting the assay,and/or a buffer, such as an assay buffer or a wash buffer, either one ofwhich can be provided as a concentrated solution, a substrate solutionfor the detectable label (e.g., an enzymatic label), or a stop solution.Preferably, the kit comprises all components, i.e., reagents, standards,buffers, diluents, etc., which are necessary to perform the assay. Theinstructions also can include instructions for generating a standardcurve.

The kit may further comprise reference standards for quantifying cTnI.The reference standards may be employed to establish standard curves forinterpolation and/or extrapolation of cTnI concentrations. In someembodiments, the reference standards for cTnI can correspond to the 99thpercentile derived from a healthy reference population. Such referencestandards can be determined using routine techniques known in the art.

Any antibodies, which are provided in the kit, such as recombinantantibodies specific for cTnI, can incorporate a detectable label, suchas a fluorophore, radioactive moiety, enzyme, biotin/avidin label,chromophore, chemiluminescent label, or the like, or the kit can includereagents for labeling the antibodies or reagents for detecting theantibodies (e.g., detection antibodies) and/or for labeling the analytes(e.g., cTnI) or reagents for detecting the analyte (e.g., cTnI). Theantibodies, calibrators, and/or controls can be provided in separatecontainers or pre-dispensed into an appropriate assay format, forexample, into microtiter plates,

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays,

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine, whole blood, plasma, or serum sample). Whereappropriate, the kit optionally also can contain reaction vessels,mixing vessels, and other components that facilitate the preparation ofreagents or the test sample. The kit can also include one or moreinstrument for assisting with obtaining a test sample, such as asyringe, pipette, forceps, measured spoon, or the like.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper,disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of traumatic brain injury or disorder.

a. Adaptation of Kit and Method

The kit (or components thereof), as well as the method for assessing ordetermining the concentration of cTnI in a test sample by an immunoassayas described herein, can be adapted for use in a variety of automatedand semi-automated systems (including those wherein the solid phasecomprises a microparticle), as described, e.g., U.S. Pat. No. 5,063,081,U.S. Patent Application Publication Nos. 2003/0170881, 2004/0018577,2005/0054078, and 2006/0160164 and as commercially marketed e.g., byAbbott Laboratories (Abbott Park, Ill.) as Abbott Point of Care (i-STAT®or i-STAT Alinity, Abbott Laboratories) as well as those described inU.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed,e.g., by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT® or theseries of Abbott Alinity devices.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., analyte antibody orcapture antibody) is attached (which can affect sandwich formation andanalyte reactivity), and the length and timing of the capture,detection, and/or any optional wash steps. Whereas a non-automatedformat such as an ELISA may require a relatively longer incubation timewith sample and capture reagent (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT® and any successor platform,Abbott Laboratories) may have a relatively shorter incubation time(e.g., approximately 18 minutes for ARCHITECT®). Similarly, whereas anon-automated format such as an ELISA may incubate a detection antibodysuch as the conjugate reagent for a relatively longer incubation time(e.g., about 2 hours), an automated or semi-automated format (e.g.,ARCHITECT® and any successor platform) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT® andany successor platform).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits, and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems. Asmentioned previously, the present disclosure is, for example, applicableto the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories)electrochemical immunoassay system that performs sandwich immunoassays.Immunosensors and their methods of manufacture and operation insingle-use test devices are described, for example in, U.S. Pat. No.5,063,081, U.S. Patent App. Publication Nos. 2003/0170881, 2004/0018577,2005/0054078, and 2006/0160164, which are incorporated in theirentireties by reference for their teachings regarding same.

In particular, with regard to the adaptation of an assay to the i-STAT®system, the following configuration is preferred. A microfabricatedsilicon chip is manufactured with a pair of gold amperometric workingelectrodes and a silver-silver chloride reference electrode. On one ofthe working electrodes, polystyrene beads (0.2 mm diameter) withimmobilized capture antibody are adhered to a polymer coating ofpatterned polyvinyl alcohol over the electrode. This chip is assembledinto an i-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the silicon chip, there is a specificbinding partner for cTnI, such as one or more cTnI antibodies one ormore monoclonal/polyclonal antibody or a fragment thereof, a variantthereof, or a fragment of a variant thereof that can bind cTnI) or oneor more anti-cTnI DVD-Igs (or a fragment thereof, a variant thereof, ora fragment of a variant thereof that can bind cTnI), any of which can bedetectably labeled. Within the fluid pouch of the cartridge is anaqueous reagent that includes p-aminophenol phosphate.

In operation, a sample from a subject suspected of suffering from TBI isadded to the holding chamber of the test cartridge, and the cartridge isinserted into the i-STAT® reader. A pump element within the cartridgepushes the sample into a conduit containing the chip. The sample isbrought into contact with the sensors allowing the enzyme conjugate todissolve into the sample. The sample is oscillated across the sensors topromote formation of the sandwich of approximately 2-12 minutes. In thepenultimate step of the assay, the sample is pushed into a waste chamberand wash fluid, containing a substrate for the alkaline phosphataseenzyme, is used to wash excess enzyme conjugate and sample off thesensor chip. In the final step of the assay, the alkaline phosphataselabel reacts with p-aminophenol phosphate to cleave the phosphate groupand permit the liberated p-aminophenol to be electrochemically oxidizedat the working electrode. Based on the measured current, the reader isable to calculate the amount of cTnI in the sample by means of anembedded algorithm and factory-determined calibration curve.

The methods and kits as described herein necessarily encompass otherreagents and methods for carrying out the immunoassay. For instance,encompassed are various buffers such as are known in the art and/orwhich can be readily prepared or optimized to be employed, e.g., forwashing, as a conjugate diluent, and/or as a calibrator diluent. Anexemplary conjugate diluent is ARCHITECT® conjugate diluent employed incertain kits (Abbott Laboratories, Abbott Park, Ill.) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.), which comprises a buffer containingMES, other salt, a protein blocker, and an antimicrobial agent.Additionally, as described in U.S. Patent Application No. 61/142,048filed Dec. 31, 2008, improved signal generation may be obtained, e.g.,in an i-STAT® cartridge format, using a nucleic acid sequence linked tothe signal antibody as a signal amplifier.

While certain embodiments herein are advantageous when employed toassess disease, such as traumatic brain injury, the assays and kits alsooptionally can be employed to assess cTnI in other diseases, disorders,and conditions as appropriate.

The method of assay also can be used to identify a compound thatameliorates diseases, such as traumatic brain injury. For example, acell that expresses cTnI can be contacted with a candidate compound. Thelevel of expression of cTnI in the cell contacted with the compound canbe compared to that in a control cell using the method of assaydescribed herein.

The present disclosure has multiple aspects, illustrated by thefollowing non-limiting examples.

17. Examples

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the presentdisclosure described herein are readily applicable and appreciable, andmay be made using suitable equivalents without departing from the scopeof the present disclosure or the aspects and embodiments disclosedherein. Having now described the present disclosure in detail, the samewill be more clearly understood by reference to the following examples,which are merely intended only to illustrate some aspects andembodiments of the disclosure, and should not be viewed as limiting tothe scope of the disclosure. The disclosures of all journal references,U.S. patents, and publications referred to herein are herebyincorporated by reference in their entireties.

The present disclosure has multiple aspects, illustrated by thefollowing non-limiting examples.

Example 1 Study 1—TBI Population

Study 1 was a large and complex project. Its institutional andpublic-private partnership is comprised of over 11 clinical sites, 7Cores, for a total of nearly 50 collaborating institutions,corporations, and philanthropy. An earlier Pilot study, based onclinical data from three clinical sites, helped refine TBI Common DataElements and created a prototype of the TBI Information Commons forStudy 1.

Subject Groups:

A total of 2,700 to 3000 TBI patients were enrolled evenly across 3clinical groups, differentiated by clinical care path: 1. Patientsevaluated in the Emergency Department and discharged (ED); 2. Patientsadmitted to the hospital, but not to ICU (ADM); and 3. Patients admittedto the ICU (ICU). An additional 100 patients per clinical group (n=300)with extracranial trauma but no TBI were enrolled as controls for atotal enrollment of 3000 patients. This stratification plan facilitatedcomparative effectiveness research (CER) analysis and was notconstrained by traditional differentiation into “Mild/Moderate/Severe”TBI. Data collection was dependent on the clinical care path (ED, ADM,ICU) and requirements of each aim. Patients in each group werestratified into 3 cohorts that define the extent of data to becollected.

The controls were adult orthopedic trauma patients who met the followingcriteria: 1. An Abbreviated Injury Score of ≤4 (not life threatening)for their extremity and/or pelvis injury and/or rib fracture; 2. Met thesame inclusion and exclusion criteria as the TBI subjects except thatthe criterion of having undergone a CT or MRI in the ED for suspectedhead injury did not apply. TBI was ruled out for the current injury byinterviewing potential controls about loss of consciousness (LOC),disturbance of consciousness, and posttraumatic amnesia (PTA)/RA; 3.Each site was provided a plan for the number of controls to targetaccording to age and gender distributions derived from the TBI Cohort;and 4. Controls were enrolled into the CA-MRI cohort for follow-up anddrop to comprehensive assessment (CA) at 2-weeks if unable to completethe MRI visit.

Subject Eligibility:

Adult patients were enrolled of all ages presenting to the EmergencyDepartment (ED) with a history of acute TBI as per American Congress ofRehabilitation Medicine (ACRM) Criteria, in which the patient hadsustained a traumatically induced physiological disruption of brainfunction, as manifested by ≥one of the following: any period of loss ofconsciousness (LOC); any loss of memory for events (e.g., amnesia)immediately before or after the accident; any alteration of mental stateat the time of the accident (feeling dazed, disoriented, and/orconfused); and/or focal neurologic deficits that may or may not bepermanent. Traumatically induced included the head being struck, thehead striking an object, or the brain undergoing anacceleration/deceleration movement (e.g., whiplash) without directexternal trauma to the head.

The Inclusion/Exclusion Criteria used is shown in Table 2.

TABLE 2 Data Criterion Source Comments Inclusion Criteria 1. Age 0-100Chart 2. Documented/verified TBI (ACRM Chart, Criteria) Interview 3.Injury occurred <24 hours ago Chart, Interview 4. Acute brain CT forclinical care Chart Subject must have brain CT scan 5. Visualacuity/hearing adequate for Chart, testing Interview 6. Fluency inEnglish or Spanish Chart, Test battery or personnel availabilityInterview 7. Ability to provide informed consent Interview ExclusionCriteria 1. Significant polytrauma that would Chart Significant bodytrauma may confound interfere with follow-up and outcome TBI outcomestesting. assessment 2. Prisoners or patients in custody Chart, Interview3. Pregnancy in female subjects Chart, Interview 4. Patients onpsychiatric hold (e.g., Chart 5150, 5250) 5. Major debilitating baselinemental Chart, Debilitating psychiatric disorders can health disorders(e.g., schizophrenia or Interview significantly impact the reliabilityof bipolar disorder) that would interfere follow up and/or posedifficulties in with follow-up and the validity of attributing to indexTBI. outcome assessment 6. Major debilitating neurological Chart,Documented debilitating baseline disease (e.g., stroke, CVA, dementia,Interview cognitive impairment will confound tumor) impairing baselineawareness outcome assessment in addition to not cognition or validity offollow-up and being fully consentable. outcome assessment 7. Significanthistory of pre-existing Chart, conditions that would interfere withInterview follow-up and outcome assessment (e.g., substance abuse,alcoholism, HIV/AIDS, major transmittable diseases that may interferewith consent, end- stage cancers, learning disabilities, developmentaldisorders) 8. Contraindications to MRI (for MRI CA + MRI cohort)Screening 9. Low likelihood of follow-up (e.g., Interview participant orfamily indicating low interest, residence in another state or country,homelessness or lack of reliable contacts) 10. Current participant in anChart, Exception to co-enrollment exclusion is interventional trial(e.g., drug, device, Interview made for sites participating inbehavioral) Resuscitation Outcomes Consortium Prehospital TranexamicAcid for TBI Study. 11. Penetrating TBI Chart 12. Spinal cord injurywith ASIA score Chart of C or worse

For each of the 3 clinical groups (i.e., ED, ADM, and ICU), the subjectswere further placed into one of three different assessment cohorts:Brief Assessment (BA Cohort), Compressive Assessment (CA) Cohort, orComprehensive Assessment+MRI (CA+MRI) Cohort. See Table 3 for Milestoneplan with 80% follow up rate.

TABLE 3 Year 1 Year 2 (Add) CA + CA + Year 3 Year 4 Total Group MRI CA NMRI CA N CA BA N BA N ED 150 87 237 50 58 108 155 100 255 300 900 ADM150 87 237 50 58 108 155 100 255 300 900 ICU 150 87 237 50 58 108 155100 255 300 900 Controls 0 99 99 0 66 66 135 0 135 0 300 Total 450 360810 150 240 390 600 300 900 900 3000

The Brief Assessment (BA) Cohort included 1200 total subjects, with 400subjects each for ED, ADM, and ICU Groups. The following data wasgathered for the BA Cohort: demographic and full clinical course data;blood draw for serum, plasma, DNA and RNA on Day 1 (<24 hours ofinjury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);clinical brain CT scan from Day 1 acquired as part of hospital course;and outcome data collected via structured telephone interview at 2weeks, 3, 6, and 12 months using NIH TBI-CDEs v.2.0 Core outcomemeasures as published on the NINDS CDE website.

The Compressive Assessment (CA) Cohort included 1200 total subjects,with 300 subjects+100 controls each for ED, ADM, and ICU Groups. Thefollowing data was gathered for the CA Cohort: demographic and fullclinical course data; high density daily clinical data for ADM and ICUGroups; blood draw for serum, plasma, RNA, and DNA on Day 1 (<24 hoursof injury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);blood draw for serum, plasma and RNA of Day 3 (48-72 hours) and 5(96-120 hours) for ADM and ICU; collection of cerebrospinal fluid ondays 1 through 5 (optional for sites to include this component); allclinical brain CT scans acquired as part of hospital course; blood drawfor serum, plasma and RNA at 2 weeks and 6 months; and outcome datacollected via structured in-person interview at 2 weeks, 6, and 12months and at 3 months via structured telephone interview using NIHTBI-CDEs v.2.0 Core, Basic and Supplemental outcome measures.

The Comprehensive Assessment+MRI (CA+MRI) Cohort included 600 totalsubjects, with 200 each for ED, ADM, and ICU Groups. The following datawas gathered for the CA+MRI Cohort: demographic and full clinical coursedata; high density daily clinical data for ADM and ICU Groups; blooddraw for serum, plasma, RNA, and DNA on Day 1 (<24 hours of injury);repeat blood draw for serum within 3-6 hours of the Day 1 baselinecollection (optional for sites to include this component); blood drawfor serum, plasma, and RNA on Day 3 (48-72 hours) and 5 (96-120 hours)for ADM and ICU; collection of cerebrospinal fluid on days 1 through 5(optional for sites to include this component); all clinical head CTscans acquired as part of hospital course; blood draw for serum, plasmaand RNA at 2 weeks and 6 months; 3T research MRI acquired at 2 weeks and6 months; and outcome data collected via structured in-person interviewat 2 weeks, 6, and 12 months and at 3 month via structured telephoneinterview using NIH TBI-CDEs v.2.0 Core, Basic, and Supplemental outcomemeasures.

Upon enrollment, data collection began in the hospital. For CA+MRIpatients, the 2-week MRI was completed at 14 days±4 days from the dateof injury. Corresponding 2-week outcomes were completed±3 days of the2-week MRI. For CA and BA patients, 2-week outcomes were completed±4days of 14 days from the date of injury. Outcomes at 3 months werecompleted±7 days of 90 days from the date of injury. For CA+MRIpatients, MRIs at 6 months were completed±14 days of 180 days from thedate of injury, with corresponding 6-month outcomes±14 days of the6-month MRI. For CA and BA patients, 6-month outcomes were completed±14days of 180 days from the date of injury. BTACT should be completed with±7 days of Outcomes (but not on the same day and no greater than 201days from injury). Outcomes at 12 months were completed±30 days of 360days from the date of injury.

hsTnI was measured in a small sample size of 59 patients from Study 1using the Abbott Architect STAT hsTnI assay (Table 4).

TABLE 4 Subject Characteristics by CT Scan and MRI Result Subject TotalCT or MRI Positive* CT or MRI Negative* Characteristics (n = 59) (n =46, 77.97%) (n = 13, 22.03%) P value Age 46.0 [24.0 to 60.0] 45.5 [23.0to 60.0] 50.0 [39.0 to 57.0] 0.7419 Sex Male 50/59 (85%) 39/46 (85%)11/13 (85%) 1.000 Female 9/59 (15%) 7/46 (15%) 2/13 (15%) Race/EthnicityAfrican-American or 6/58 (10%) 4/45 (9%) 2/13 (15%) 0.2398 AfricanCaucasian 48/58 (83%) 39/45 (87%) 9/13 (69%) Hispanic 4/58 (7%) 2/45(4%) 2/13 (15%) TBI History Yes, with No LOC 9/56 (16%) 3/43 (7%) 6/13(46%) 0.0037 Yes, with LOC 8/56 (14%) 6/43 (14%) 2/13 (15%) No Prior TBI39/56 (70%) 34/43 (79%) 5/13 (38%) ED Presentation Loss of ConsciousnessNo 6/58 (10%) 2/45 (4%) 4/13 (31%) 0.0227 Yes 47/58 (81%) 38/45 (84%)9/13 (69%) Unknown 5/58 (9%) 5/45 (11%) Glasgow Coma Scale 15.0 [3.0 to15.0] 14.0 [3.0 to 15.0] 15.0 [15.0 to 15.0] 0.0162 Glasgow Coma ScaleClassification Severe (3-8) 16/59 (27%) 16/46 (35%) 0.0177 Moderate(9-12) 3/59 (5%) 3/46 (7%) Mild (13-15) 40/59 (68%) 27/46 (59%) 13/13(100%) Mechanism of Injury Motor Vehicle 10/59 (17%) 9/46 (20%) 1/13(8%) 0.2975 (driver/passenger) Motorcycle/ATV/golf cart 5/59 (8%) 3/46(7%) 2/13 (15%) (driver/passenger) Individual struck by any 3/59 (5%)2/46 (4%) 1/13 (8%) type of vehicle Fall from a moving object 3/59 (5%)3/46 (7%) (bike/skateboard/horse/etc.) Fall from stationary object 27/59(46%) 20/46 (43%) 7/13 (54%) (roof/latter/etc.) Assault 10/59 (17%) 9/46(20%) 1/13 (8%) Struck on head by object, 1/59 (2%) 1/13 (8%) notassault (tree/etc.) Alcohol Level (g/dL) 0.1 [0.0 to 0.2] 0.1 [0.0 to0.2] 0.0 [0.0 to 0.0] 0.1588 Drug Screen Negative 51/59 (86%) 41/46(89%) 10/13 (77%) 0.3567 Positive 8/59 (14%) 5/46 (11%) 3/13 (23%)Biomarker Results Collection Time Since 771.0 (+/−339.8) 779.4(+/−296.8) 743.0 (+/−468.7) 0.7383 Injury (Minutes) Prognostic ScoresGlasgow Outcome Scale 6.0 [5.0 to 7.0] 5.5 [4.0 to 7.0] 7.0 [7.0 to 7.0]0.0130 (3 months) Glasgow Outcome Scale 6.0 [5.0 to 7.0] 6.0 [4.0 to7.0] 7.0 [5.5 to 7.5] 0.1941 (6 months) Glasgow Outcome Scale 7.0 [5.0to 8.0] 6.5 [5.0 to 8.0] 7.0 [6.0 to 8.0] 0.4412 (12 months) RivermeadQuestionnaire 0.0 [0.0 to 2.0] 0.0 [0.0 to 2.5] 0.0 [0.0 to 2.0] 0.8378First 3 Items (6 months) Rivermead Questionnaire 9.0 [4.0 to 15.0] 8.5[4.0 to 15.0] 13.0 [0.0 to 27.0] 0.5449 Last 13 Items(6 months) WAIS-IIIProcessing 30.0 [5.0 to 55.0] 30.0 [5.0 to 50.0] 43.0 [18.0 to 77.0]0.3235 Speed Index (6 months) Satisfaction with Life 21.5 (+/−6.2) 21.7(+/−5.7] 20.4 (+/−8.5) 0.6205 Scale (6 months) Functional Independence126.0 [125.0 to 126.0] 126.0 [124.0 to 126.0] 126.0 [126.0 to 126.0]0.2958 Measure (6 months) *24 subjects received an MRI. Continuousvariables are presented as median [25-75% Inter Quartile Range] andcompared using Wilcoxon rank sum test or Mean (+/−SD) and compared usinga t-test based on the distribution of the data. Categorical variablesare presented as number/total (percent) and compared using Chi-Square orFisher's exact test.

In addition to a blood draw within 24 hours of brain injury, eachpatient had an extensive medical evaluation including head CT,neuropsychiatric testing, Glasgow Coma Score (GCS), and many patientsalso had a follow up MRI within 2 weeks of injury. Following ameticulous standardized blood draw protocol and processing, plasmasamples were aliquotted for storage at −80° C., later thawed and tested.Each sample was run in duplicate with the listed results being anaverage of the two runs. Table 4 shows that ethanol (ETOH) levels didnot correlate with biomarker levels (Pearson Correlation=0.023,p-value=0.89) as ETOH consumption is frequently related to TBI, inparticular severe TBI.

Table 5 shows the analysis of data in a small sample size of 191patients from Study 1, specifically, the analysis of the change betweenTime Point 2 and Time Point 1 in hsTnI levels. The Time Point 1 sampleis taken within 24 hours from injury and Time Point 2 sample is takenabout 3-6 hours after the Time Point 1 sample is taken. HsTnI appears tohave a significant change from Time Point 1 to Time Point 2 (n=89,median delta (i.e., change from Time Point 1 to Time Point 2)=0.091537pg/mL, Wilcoxon Signed-Rank Test p-value<0.6085). See also FIG. 1. FIG.1 shows a box and whisker plot indicating the hsTnI levels determined inall of the patients of the study at Time Point 1 and Time Point 2. Thesedata were simply analyses comparing the time points, and are not basedon CT, MRI or GCS+/−.

TABLE 5 Sam- Wilcoxon ple Signed- As- Size Median Minimum Maximum Rank Psay (n) Delta Delta Delta Statistic value hsTnI 89 0.091537 −226.483729419.421 122 0.6085

The data from the 191 samples from Study 1 was further analyzed based onwhen the Time Point 1 sample was taken, e.g., 0 to about 6 hours afterinjury (“0-6 hour group”), about 6 to about 12 hours after injury (“6-12hour group”) and correlated with head CT scan results and/or GCS scores.The hsTnI levels in the Time Point 1 and Time Point 2 samples werecompared to the positive or negative head CT scan results and/or GCSscores indicating mild or moderate/severe TBI. As this analysis wasbased on the time of when the Time Point 1 sample was taken, the numberof subjects per group was small. See Table 6.

TABLE 6 Time Range (hours)* Total # of 0-6 6-12 12-18 18-24 >24 SubjectsCT Scan Positive 6 21 33 31 4 95 Negative 8 17 25 25 0 75 GCS Mild 12 2745 41 1 126 Score Moderate/ 2 10 15 15 2 44 Severe *time of first sampletaken from time of injury

CT Scan.

FIG. 2 shows the hsTnI results in the subjects from the 0-6 hour groupthat had positive or negative head CT scans. FIG. 2 shows thedistribution of the hsTnI levels at Time Point 1 and Time Point 2 in thesubjects that had a positive or negative CT scan. The hsTnI levels inthe CT positive subjects were higher than the CT negative subjects atboth Time Point 1 and Time Point 2 for the 0-6 hour group. The hsTnIlevels in the subjects from the 0-6 hour group having a positive CT scanhad a striking increase from Time Point 1 to Time Point 2.

FIG. 3 shows the absolute amount (“absolute delta”) in hsTnI levels insubjects from the 0-6 hour group. Subjects having a positive head CTscan had a greater change in hsTnI levels from Time Point 1 to TimePoint 2 compared to subjects having a negative head CT scan.

GCS Scores.

FIG. 4 shows the hsTnI results in the subjects from the 0-6 hour groupthat were identified as having mild or moderate to severe(“moderate/severe”) TBI based on their GCS score. FIG. 4 shows thedistribution of the hsTnI levels at Time Point 1 and Time Point 2 in thesubjects determined to have mild or moderate/severe. The hsTnI levels inthe moderate/severe subjects were significantly higher than the mildsubjects at both Time Point 1 and Time Point 2 for the 0-6 hour group.The hsTnI levels in the 0-6 hour group had a striking increase from TimePoint 1 to Time Point 2.

FIG. 5 shows the absolute amount (or change between Time Point 1 andTime Point 2; “absolute delta”) in hsTnI levels of 0-6 hour group.Subjects determined to have moderate/severe TBI based on the GCS scorehad a larger change in hsTnI levels from Time Point 1 to Time Point 2compared to subjects determined to have mild TBI based on the GCS score.

Example 2 Study 2—Development of a Multi-Modality Classification Schemefor Traumatic Brain Injury

The goal of this study was to develop a classification scheme for braininjury that indicates the nature (type) and severity of injury. Forexample, serum biomarkers revealed cell type. Trauma patients weredivided into three groups for analysis: only brain injured, onlynon-brain injured, and combined injury. Brain injured and non-braininjured trauma groups were compared to each other and to the combinationof brain/nonbrain trauma. These trauma groups were compared tonon-trauma controls. CSF from trauma patients was compared to CSF fromnon-trauma patients. A secondary goal was to determine whether any ofthe measures, alone or in combination, had utility as a predictor ofclinical outcome after TBI.

An objective multi-modality classification scheme and outcome measurefor traumatic brain injury was developed based on several measures: 1)blood-based biomarkers; 2) physiologic measures and evaluation; and 3)radiographic measures (CT and 3T MRI). Blood-based biomarkers canindicate which cell types are damaged (e.g., glial vs. neuronal) andradiography can detect structural changes.

Study Site:

Trauma patient were recruited at Hennepin County Medical Center (HCMC)in the state of Minnesota. Participants included trauma patients of allages presenting to the HCMC Emergency Department (ED), trauma bay, or asdirect transfer to neurosurgery. Trauma patients were excluded if theyhad a major psychiatric or neurologic disorder, were developmentallyabnormal, or were prisoners. Subjects were identified by searchingmedical records for all trauma admissions and cross-checking with theAmerican College of Surgeons trauma registry utilized in the hospital.

All trauma patients were recruited for screening at the time ofpresentation and underwent: 1) a standardized (templated) history andphysical examination; 2) analysis of serum biomarkers if blood is drawnfor other indications; 3) radiographic study as clinically indicated; 4)follow-up as clinically indicated with 1)-3); 5) pathologic specimenanalysis in patients going to the operating room only; 6) CSF analysisin patients receiving ventriculostomy catheters only; 7) brain tissueoxygenation analysis in patients receiving Licox only; and 8) outcomeassessment in the TBI center as clinically indicated. At the time ofadmission, the potential participants underwent a 24 hour screeningprocess (Table 7) before providing informed consent.

TABLE 7 Screening Assessments Adult Pediatric All Surgery-Trauma Historyand Physical Selections from Neurosurgery-Trauma History and PhysicalAwake SCAT3: SAC, SSS-C Child SCAT3: SAC-C, SSS-C OR Pathogenic SpecimenVC CSF Analysis Licox Brain Tissue Oxygenation OR: Patients going to theoperating room; VC: Patients receiving ventriculostomy catheters; Licox:Patients receiving Licox

In addition, patients who consented and controls (age and gendermatched) undergone the above plus the following additional studies: 1genomic, serum, and CSF; and 2) 3T MRI in select circumstances (serummarkers in the test group; normal markers in the control group). Traumapatients included the full spectrum ranging from non-brain injured,CT-negative to structurally brain injured, requiring surgery. A patientsand controls were recruited over approximately 15 months. Survivingtrauma subjects were followed until they were discharged from HCMCservices. Subjects evaluated in the ER and released were invited forresearch follow-up.

The screening process included a standardized and templated medicalhistory and physical examination. The template was the current“Surgery-Trauma History and Physical” template in EPIC with oneadditional question that asks if the patient suffered a head trauma. Ifthe patient did, three sections automatically dropped down foradditional information. The first was information from the Neurosurgerytrauma history and physical template, including subarachnoid grade,hemorrhage grade, intracerebral hemorrhage, and social history (level ofeducation, employment, living arrangements, and ethnicity). The finaltwo were standardized brain injury assessment tools: the StandardizedAssessment of Concussion (SAC) and the Symptom Severity Score (SSS).Pediatric versions of these assessments were available and used whenindicated. Additionally, the loss of consciousness question alreadyincluded in the “Surgery Trauma History and Physical” template wascopied into this drop down section with a subset of questions thatprovided a more clear understanding of the loss of consciousness eventand the patient's current orientation. The most clinically accurateassessment taken during the first 24 hours of admission was used forfuture data analysis.

Non-TBI subjects were also included based on the following criteria: bebetween 15 and 50 years of age at the time of enrollment; have similarcharacteristics as the TBI population in terms of gender, age,handedness, educational level, and scanner criteria; and be capable ofsufficiently clear communication and language fluency to allow thesubject to provide written informed consent, or assent with parental orguardian consent for minors, and to complete study assessments forparticipation in all parts of the study. Non-TBI subjects were excludedif they have: been diagnosed of mild TBI within the past 6 months; aprior moderate to severe TBI (GCS<13) within past 10 years; epilepsywith recurring seizures in the past 10 years; drug abuse (exceptmarijuana) in the past 10 years based on DAST-10 screening; alcoholabuse based on AUDIT-C screening; current primary Axis I or IIpsychiatric disorders, except for disorders classified as minor and notexpected to impact study conduct or integrity; a history of brain mass,neurosurgery, stroke, white matter disease, and/or dementia; a knowncognitive dysfunction or structural brain disease/malformation; astructural brain injury on prior neuroimaging findings; been prescribedantipsychotic/antiepileptic medications; been unable (such as due tourgent medical care needs) or unwilling to complete study proceduresaccurately or have any conflict of interest that could affect studyresults, in the opinion of the investigator; or contraindications to MRIscanning, including: a. current or suspected pregnancy per sitepractice; b. other conditions that may constitute a hazard to thesubject during study participation, per investigator; and c. inabilityto comply with any part of the site's MR safety policy.

Specimen Collection and Handling.

Up to 40 mL (approximately 3 tablespoons) of blood were obtained at eachspecimen collection. 2 tubes of serum and 2 tubes of plasma were drawnat Encounters 1, 2 4, and 5. At Encounter 3, 2 tubes of serum, 1 tube ofplasma and 1 or 2 tubes of whole blood were drawn. These study specimenswere processed, aliquoted, frozen, and shipped to Abbott Laboratoriesfor biomarker testing and storage. Sample aliquots were sent to testingsites for additional TBI biomarker testing. A specimen was consideredunevaluable for the study if: it contained insufficient volume toperform necessary measurements, it was grossly hemolyzed, lipemic, oricteric; it was not collected in the proper type of collection tube; itwas not properly labeled; or it was not properly stored by thecollection site or at Abbott Laboratories.

Serum specimens were obtained via blood draw. If a blood draw wasobtained at the time of admission for clinical purposes, additionalspecimen was obtained and retained for research purposes. If blood wasnot drawn for clinical purposes, trained research personnel drawn theblood required for research. The blood was drawn through venipunctureunless a central venous access was required by the standard of care, inwhich case the blood was withdrawn via that access. The first blood drawwas taken upon admission, the second 3-6 hours after the first, and thethird was taken at 24 hours after the trauma. Discovery efforts werealso ongoing to find genetic markers of susceptibility to TBI orpredictive markers of TBI. At each time point, 40 mL (less than 3tablespoons) of blood was collected: 20 mL of serum (2 tubes) and 20 mLof plasma (2 tubes). During Encounter 1, only 2 tubes of serum and 1tube of plasma were collected for blood biomarker analysis. This wholeblood collection was 6.0 mL in a whole blood tube. If a patient wasenrolled at Encounter 2 instead of Encounter 1, they had 2 tubes ofserum and 1 tube of plasma collected for blood biomarker analysis. Thiswhole blood collection was 6.0 mL in a whole blood tube.

The amount of blood drawn was limited according to the NINOSstandardized table (Table 8). The number of attempts to draw blood waslimited for children under the age of seven to two attempts. In the casethat the NINOS standardized table did not allow enough blood to be drawnfor the study or there were two failed attempts to draw blood in a childpatient, access to leftover blood samples drawn under the clinicalstandard of care was requested for complete biomarker analysis in thisstudy. This blood draw allowed for the analysis of up to 390 blood-basedbiomarkers related to traumatic brain injuries.

TABLE 8 Maximum Allowable Total Blood Draw Volumes Minimum Hgb Maximumallowable required at time volume (mL) in Maximum of blood draw oneblood draw volume (clinical + Minimum Hgb if subject has Body Wt Body WtTotal blood (=2.5% of total research) (mL) in required at timerespiratory/ (Kg) (lbs) volume (mL) blood volume) a 30-day period ofblood draw CV compromise 1 2.2 100 2.5  5 7.0 9.0-10.0 2 4.4 200 5 107.0 9.0-10.0 3 6.3 240 6 12 7.0 9.0-10.0 4 8.8 320 8 16 7.0 9.0-10.0 511 400 10 20 7.0 9.0-10.0 6 13.2 480 12 24 7.0 9.0-10.0 7 15.4 560 14 287.0 9.0-10.0 8 17.6 640 16 32 7.0 9.0-10.0 9 19.8 720 18 36 7.0 9.0-10.010  22 800 20 40 7.0 9.0-10.0 11-15 24-33  880-1200 22-30 44-60 7.09.0-10.0 16-20 35-44 1280-1600 32-40 64-80 7.0 9.0-10.0 21-25 46-551680-2000 42-50  64-100 7.0 9.0-10.0 26-30 57-66 2080-2400 52-60 104-1207.0 9.0-10.0 31-35 68-77 2480-2800 62-70 124-140 7.0 9.0-10.0 36-4079-88 2880-3200 72-80 144-160 7.0 9.0-10.0 41-45 90-99 3280-3600 82-90164-180 7.0 9.0-10.0 46-50 101-110 3680-4000  92-100 184-200 7.09.0-10.0 51-55 112-121 4080-4400 102-110 204-220 7.0 9.0-10.0 56-60123-132 4480-4800 112-120 224-240 7.0 9.0-10.0 61-65 134-143 4880-5200122-130 244-260 7.0 9.0-10.0 66-70 145-154 5280-5600 132-140 264-280 7.09.0-10.0 71-75 156-165 5680-6000 142-150 284-300 7.0 9.0-10.0 76-80167-176 6080-6400 152-160 304-360 7.0 9.0-10.0 81-85 178-187 6480-6800162-170 324-340 7.0 9.0-10.0 86-90 189-198 6880-7200 172-180 344-360 7.09.0-10.0 91-95 200-209 7280-7600 182-190 364-380 7.0 9.0-10.0  96-100211-220 7680-8000 192-200 384-400 7.0 9.0-10.0

In addition to the initial physical exam, those patients that were sentto the operating room undergone pathologic specimen analysis, thosepatients that received Licox had brain tissue oxygenation informationrecorded, and those patients that received ventriculostomy catheters hadCSF collected for analysis. In order to analyze the CSF, 5.0 mL wascollected at the same intervals that blood was drawn. Radiographicstudies were performed in accordance with the standard of care. None ofthe assessments performed during the screening processes were analyzedwith the rest of the data until informed consent was obtained. If thepatient did not ultimately consent to research, the specimens, andinitial assessments were discarded.

After the participants were discharged, the patients' medical recordswere accessed for information about the clinical course, including timespent in the ED, any surgeries or other neuromonitoring methods used,and the acute care outcome evaluation. If the patient spent time in theICU, information was extracted from that time period as well, includingdata from Moberg monitors and daily therapeutic intensity level.

Trauma patients were divided into three groups for analysis: only braininjured, only non-brain injured, and combined injury. Two age- andgender-matched control groups were included in this study and recruitedfrom the ED: non-trauma and CSF controls. Non-trauma controls were thosewho did not experience any trauma, and this group composed largely offamily and friends of the patients admitted for brain injury. Bothcontrol groups were consented to undergo a single intensive assessmentthat included a blood draw and cognitive, neurological, and quality oflife assessments (SAC, NOS-TBI, QoLABI). Patients receiving electiveventriculostomy or lumbar drain catheters were (pre-operatively)consented to be a part of the CSF control group. 5 mL of CSF wascollected from the ventriculostomy catheter of patients in this controlgroup for comparison with the CSF collected from the portion of thestudy group that received a ventriculostomy catheter as a part of theirstandard of care. The CSF control group was also offered the chance toparticipate in the same intensive assessment as the other two controlsgroups that included a blood draw and a 3T MRI scan.

Follow-Up:

All patients that consented to participate in the follow-up portion ofthe study were asked to return to the hospital. Patients who returnedwere seen in the TBI Outpatient Clinic at 2 weeks, 4 weeks, months, 6months, and 1 year. If they did not have a scheduled appointment at theTBI Outpatient Clinic, they were scheduled a time to come into the BrainInjury Research Lab (PL.610) at those time points. Table 9 provides atimeline for each of the assessments. Blood draws for biomarker analysiswere done at each of the five follow-up time points in the same method,as described above. The outcome assessment battery listed in Tables 10and 11 were completed at 3 months, 6 months, and one year. Radiographicscans that were a part of the standard of care were accessed through theparticipant's medical records, but select consented participants andcontrols also underwent 3T MRI scans at 2 weeks and 6 months after theirbrain injury. Each MRI examination took approximately one hour andincluded the following pulse sequences: (1) Sagittal short TR localizer,(2) Axial Fse, (3) Axial FLAIR, (4) Axial SWI, (5) Axial T2* imaging. Inthe case that patients were not able to come into the hospital forfollow-up, they were contacted via phone at three months and one yearafter their injury to complete the BT ACT, which was a 15-20 minutecognitive assessment designed to be administered over the phone.

TABLE 9 Outcome Timeline Blood 3T CSF CT Assess- Total Time Draw MRICollection Scan ments (minutes) ADM X X (if 10 indicated) 2 weeks X X 704 weeks X 10 3 months X X 70 6 months X X 70 1 year X X X* X 160 (130without CT)

TABLE 10 Outcome Assessments - Not Finalized Adult Pediatric Out- AllGOS and GOSE Pediatric GOSE come Awake SCAT3: SSS and Child SCAT3: Child& Parent SAC Report; SAC-C GOAT COAT Duration of Duration of AmnesiaAmnesia NOS-TBI NOS-TBI Quality of Life: Quality of life: MPAI-4Neuropsychiatric Rating Schedule Pediatric Quality of Life Inventory(For Child and for Proxy) Not CRS-R just Brain Stem Reflex Grid?) Awake

TABLE 11 Possible Infant Assessments Name Age Time Description BayleyIII,  0-3.5 30-90 Cognitive, language (receptive and BSID* expressive)and motor development Most commonly used in test for this age rangeBITSEA* 1-3  7-12 Parent perception of Social and Emotional behavior 17items of 42 are for autism, so may be able to be made shorter CBCL1.5-5  25-30 Parent perception of performance on Activities, Social, andSchool performance MSEL* 1 15 Cognitive and Motor Ability (Gross 3 25-35Motor, Visual Reception, Fine Motor, 5 40-60 Language Mostly forreadiness for school Shape 3-6 45-75 Inhibition and Switching Processes:School* Emerging Executive Functions Trails- 2-6  5-10Neuropsychological Function: Psycho- Preschool* motor speed, ComplexAttention, Exec- utive Function Advanced Trail Making Test *RequestedAccess

Statistical Analysis Plan.

Biomarker data was analyzed by examining maximum concentration draw foreach biomarker per patient, or by time from incident buckets, or both.To address the primary objective of determining associations betweenbiomarker concentrations in blood and clinical neurological and magneticresonance imaging data, multiple analyses was employed. Principalcomponents analysis was used to examine which biomarkers may beexplaining the same variance, or if a biomarker was contributing verylittle variance. The biomarkers were used in a logistic regressionanalysis, and some biomarkers were excluded based on the results fromthe principal components analysis, and clinical input. A significancelevel of 0.05 was used for the logistic regression analysis. ROCanalysis was also used to examine the predictive ability of eachbiomarker in determining MRI status or neurological testing outcomes,for this set of data.

Example 3 Study 2—Analysis

High sensitivity troponin I (hsTnI) was measured using the AbbottArchitect STAT hsTnI assay in samples taken from subjects, as describedin Example 5. FIGS. 6 and 7 show that hsTnI levels correlated withinjury throughout the first 24 hours after injury (range approximately2-23 hours) based on CT scan results (FIG. 6) and GCS score (FIG. 7).

Samples Taken from Subjects within 2 Hours of Injury:

hsTnI levels in samples taken from human subjects within about 2 hoursof suspected injury were measured. FIG. 8 shows ROC analysis of hsTnIlevels correlated with CT status (positive vs. negative CT scan result;AUC=0.430). Table 12 shows the sensitivities and specificities of usinghsTnI cut-off levels to predict a positive CT scan result.

TABLE 12 CT Scan - hsTnI Reference Levels Analysis Biomarker Levels(pg/mL) Sensitivity (%) Specificity (%) hsTnI 1.15 87.5 31.25 1.29 75.031.25

FIG. 9 shows ROC analysis of hsTnI correlated with GCS Score results(mild vs. moderate/severe TBI; AUC=0.588). Table 13 shows thesensitivities and specificities of using hsTnI cut-off levels to predictmoderate/severe TBI based on GCS scores.

TABLE 13 GCS Score - hsTnI Reference Levels Analysis Biomarker Levels(pg/mL) Sensitivity (%) Specificity (%) hsTnI 5.80 85.71 33.33 4.7185.71 40.0

Samples Taken from all Subjects:

FIG. 10 shows the ROC curve correlating the hsTnI levels for all of thesubjects at Time Point 1 with CT scan result. The sensitivity andspecificity of reference levels for for all of the subjects based on theROC curve is shown in Table 14.

TABLE 14 Reference Level (pg/mL) Sensitivity (%) Specificity (%) 1.6581.58 20.59 2.16 71.05 30.88 14.75 31.58 79.41 30.43 21.05 82.35

FIG. 11 shows the ROC curve correlating the hsTnI levels for all of thesubjects at Time Point 1 with GCS scores. The sensitivity andspecificity of reference levels for 0-6 hour group based on the ROCcurve is shown in Table 15.

TABLE 15 Reference Level (pg/mL) Sensitivity (%) Specificity (%) 43.7932.14 94.87 21.23 46.43 85.90 1.94 85.71 30.77 2.54 75 38.46

Example 4 Study 2—Absolute Amount Analysis

Samples Taken from Subjects within 2 Hours of Injury:

hsTnI levels were measured in samples taken from human subjects at afirst time point within about 2 hours of suspected injury and samplestaken from the human subjects at a second time point 3-6 hours after thefirst time point. FIG. 12 shows ROC analysis of absolute amount(“absolute delta”) of hsTnI results (i.e., the absolute differencebetween hsTnI levels at Time Point 2 and hsTnI levels at Time Point 1)correlated with CT status (positive vs. negative CT scan result;AUC=0.514). Table 16 shows the sensitivities and specificities of usinghsTnI cut-off levels to predict a positive CT scan result, wherein avalue less than the cutoff is predicted to have a positive CT scanresult.

TABLE 16 CT Scan - hsTnI Absolute Delta Analysis Absolute AmountBiomarker (pg/mL) Sensitivity (%) Specificity (%) hsTnI 2.54 66.67 55.561.16 83.33 27.78

FIG. 13 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with GCS Scoreresult (mild vs. moderate/severe; AUC=0.380). Table 17 shows thesensitivities and specificities of using hsTnI cut-off levels to predictmoderate/severe TBI based on GCS scores, wherein a value less than thecutoff is predicted to have moderate/severe TBI.

TABLE 17 GCS Score - hsTnI Absolute Delta Analysis Absolute AmountBiomarker (pg/mL) Sensitivity (%) Specificity (%) hsTnI 4.73 66.67 31.2520.62 83.33 12.5

Samples Taken from all Subjects:

FIG. 14 shows the ROC curve correlating the hsTnI levels for all of thesubjects at Time Point 1 with CT scan result. The sensitivity andspecificity of absolute amounts (or change between Time Point 1 and TimePoint 2) in hsTnI levels for all of the subjects based on the ROC curveare shown in Table 18.

TABLE 18 Absolute amount (pg/mL) Sensitivity (%) Specificity (%) 16.05088.24 19.36 10.755 82.35 19.36 0.720 35.29 70.97 0.389 29.41 90.32

FIG. 15 shows a ROC curve correlating the absolute amount of change inhsTnI levels for all of the subjects at Time Point 1 and Time Point 2with GCS scores (mild vs. moderate/severe). The sensitivity andspecificity of the absolute amounts (or change between Time Point 1 andTime Point 2) in hsTnI levels for all of the subjects based on the ROCcurve is shown in Table 19.

TABLE 19 Absolute amount (pg/mL) Sensitivity (%) Specificity (%) 3.3345.46 32.43 5.80 72.73 21.62 17.17 81.82 13.51

Example 5 TBI Population

Recent introduction of high sensitivity troponin assays (hsTn) provide anew window of opportunity for improved characterization of cardiacinjury. hsTn assays measure up to ten-fold lower concentrations ofcardiac troponin than conventional troponin assays with improvedprecision. They allow improved detection of cardiac injury innon-cardiac conditions and therefore afford an unprecedented opportunityfor improved characterization of cardiac injury across the spectrum ofTBI severities. The objectives of this study were: to estimate theincidence cardiac injury across the spectrum of TBI with precision; toidentify the risk factors associated with cardiac injury; to determinewhether cardiac injury is associated with functional and symptomaticoutcomes post-TBI; and to characterize longitudinal changes in troponinvalues in TBI.

High sensitivity troponin I (hsTnI) was measured in samples fromsubjects using the Abbott Architect STAT hsTnI assay. HsTnI was measuredon blood samples obtained at 0, 4, 24, and 1 month after presentation.

Study Population.

A 6-month prospective cohort study of patients evaluated for TBI in theemergency department and age and gender matched traumatically injurednon-TBI control subjects. Participants were recruited from two academicEDs. Eligible TBI participants were 18 years or older and 1) presentedto the ED within 24 hours of blunt head injury, 2) met the ACEP criteriafor evaluation with a head CT scan, and 3) consented to research blooddraws. Excluded from the study were subjects with: brain tumor; severedementia; prior history of intracranial hemorrhage or intracranialsurgery; seizure induced head injury; pregnancy; no working telephonenumber; inability to communicate in English; or blood transfusion beforeinitial research blood draw was obtained. Age and gender matched traumacontrol participants were 18 years or older, presented to the ED within24 hours of traumatic injury but had no evidence of head injury, andconsented to research blood draws. Exclusion criteria for trauma controlparticipants were the same as that for TBI participants.

Clinical Data.

Demographic and clinical data were collected by trained researchcoordinators using structured data collection tools recommended by theNational Institute of Neurological Disorders and Stroke (NINdS) CommonData Elements. Data were collected and managed using REDCap electronicdata capture tools hosted at the Johns Hopkins School of Public Health.

Outcome Data.

Mild TBI (mTBI) was defined as per the American Congress ofRehabilitation Medicine (ACRM) which defines mTBI as a traumaticallyinduced physiological disruption of brain function as a consequence ofthe head being struck, striking an object, or undergoing anacceleration/deceleration movement without direct external head traumaand resulting in at least one of the following: any period of loss ofconsciousness of 30 minutes or less; any loss of memory for eventsimmediately before or after the accident (post-traumatic amnesia)lasting no more than 24 hours; any alteration in mental status at thetime of the accident (e.g., confusion, disorientation); focalneurological deficit(s); or a Glasgow Coma Scale (GCS) score of 13-15 atpresentation.

Head CT images were read by one board certified neuroradiologist. Thepresence/absence of traumatic lesions, including the size and locationof these lesions were classified according to the NINDS common dataelements for radiologic imaging of TBI (Duhaime et al., Arch Phys MedRehabil. (2010) 91:1661-1666). Outcome data were collected at 1, 3, and6 months after enrollment either via telephone interview (61.8% of theTBI population) or in-person assessment (38.2% of the population).Functional outcome was ascertained using the Extended Glasgow OutcomeScale (GOSE) which categorizes on a scale of 1 (Dead) to 8 (upper goodrecovery). Delayed functional recovery at 1 month was defined as GOSE<8.As per the International Statistical Classification of Diseases andRelated Health Problems 10th Revision (ICD-10) criteria, subjects weredeemed to have mild post-concussion syndrome (PCS) if they reported amild/moderate/severe problem in 2 of the following ICD-10 symptomscategories as measured by the Rivermead Post-Concussion Questionnaire(RPQ): (1) headaches, dizziness, general malaise, excessive fatigue, ornoise intolerance; (2) irritability, emotional lability, depression, oranxiety; (3) subjective complaints of concentration or memorydifficulty; (4) insomnia; (5) reduced tolerance to alcohol; (6)preoccupation with these symptoms and fear of permanent brain damage.Similarly subjects were deemed to have at least moderate PCS if theyreported mild/moderate/severe problems in at least 3 of the ICD-10symptom categories listed above. Moderate/severe depressive symptomswere defined as a total score of 10 or more on the Patient HealthQuestionnaire 9 (PHQ9).

Statistical Analysis.

hsTnI values between TBI and trauma control subjects were compared.Additionally, hsTnI values were compared among TBI subjects withtraumatic intracranial abnormalities (positive head CT) on head CT scan,TBI with a negative head CT who meet the ACRM criteria for TBI, and TBIsubjects with a negative CT who don't meet the ACRM criteria. Groupcomparisons of median hsTnI values were made using the Kruskall-Wallistest. Whether the location and size of intracranial lesions wereassociated with hsTnI was also determined. A multiple linear regressionmodel with generalized estimating equations (GEE) and robust varianceestimation to account for correlations within repeated measurements ofhsTnI was used to examine longitudinal changes in hsTnI measured at 0,4, 24, 72 hours, 1 week, and 1 month after presentation. Logisticregression models were constructed to determine whether hsTnI measuredat presentation was associated with functional disability (measured bythe Glasgow Outcome Scale Extended), post-concussive symptoms (measuredby the Rivermead Post-concussion symptom inventory) and depression(measured by the Patient Health Questionnaire 9) at 1, 3, and 6 monthspost-injury.

Sample Size.

The number of samples for each criterion is shown in Table 20.

TABLE 20 Time Number TBI - 0 hour 500 TBI - 4 hour 338 TBI - 24 hour 134TBI - 1 month 73 Trauma control - 0 hour 94

Example 6 hsTnI Analysis

FIG. 16 shows box plots of hsTnI levels in TBI patients (n=500) andTrauma control patients (n=94) (p=0.0001). Initial hsTnI values on theday of injury are higher in TBI patients than in controls

FIG. 17 shows box plots of hsTnI levels in TBI patients with abnormalhead CT (n=98) versus normal head CT scan (n=402) (p=0.005). InitialHsTnI values on the day of injury were higher in participants withabnormal head CTs than in those with normal head CT

FIG. 18 shows box plots of hsTnI levels in TBI patients with GCS scoresof <13 (n=9), 13 (n=8), 14 (n=54), or 15 (n=429) (p=0.46). There was nostatistically significant difference in initial hsTnI values on the dayof injury, according to GCS level.

FIG. 19 shows box plots of hsTnI levels in TBI patients having GOSEscores at 1 month post-injury of 1 (n=6), 3 (n=4), 4 (n=8), 5 (n=46), 6(n=67), 7 (n=100), or 8 (n=149) (p=0.001). Patients with severedisability or worse at 1 month i.e. GOSE of less than 5, had higherinitial hsTnI on the day of injury, than those with better functionaloutcome (i.e. GOSE of 5 or greater)

FIG. 20 shows box plot of hsTnI levels in TBI patients having GOSEscores at 3 months post-injury of 1 (n=9), 3 (n=2), 4 (n=7), 5 (n=22), 6(n=45), 7 (n=109), or 8 (n=154) (p=0.02). Initial hsTnI values on theday of injury were higher among subjects with severe disability or worseat 3 months i.e. GOSE of less than 5, had a higher initial hsTnI on theday of injury, than those with better functional outcome (i.e. GOSE of 5or greater)

FIG. 21 shows box plot of hsTnI levels in TBI patients having GOSEscores at 6 months post-injury of 1 (n=10), 2 (n=1), 3 (n=3), 4 (n=5), 5(n=17), 6 (n=41), 7 (n=102), or 8 (n=158) (p=0.11). There was a trendtowards higher initial hsTnI on the day of injury in subjects withsevere disability or worse at 3 months i.e. GOSE of less than 5, thanthose with better functional outcome (i.e. GOSE of 5 or greater). Due tothe smaller sample size at this time point, the difference approximatedby did not reach statistical significance.

FIG. 22 shows box plot of hsTnI levels in samples taken at 0 hours (n=98for abnormal; n=402 for normal), 4 hours (n=88 for abnormal; n=250 fornormal), 24 hours (n=51 for abnormal; n=83 for normal), or 1 month (n=14for abnormal; n=59 for normal) after injury from TBI patients havingabnormal head CT scan and normal head CT scan. In both groups (thosewith abnormal head CT cans and those with normal head CT scans),troponin values were elevated during the first 24 hours after injury andreturned to lower values at 1 month post-injury.

FIG. 23 shows box plot of the 4 hour absolute change in hsTnI levels inTBI patients having GOSE scores at 1 month post-injury of 1 (n=5), 3(n=4), 4 (n=8), 5 (n=31), 6 (n=45), 7 (n=71), or 8 (n=99) (p=0.004).Subjects with severe disability or worse at 1 month post-injury (i.e.GOSE less than 5) had higher 4 hour absolute changes in hsTnI levelsthan those with better functional outcome (i.e. GOSE of 5 or greater).

FIG. 24 shows box plot of hsTnI levels in TBI patients based on age: 18to 30 years, 30 to 50 years, 50 to 70 years, and >70 years (p=0.0001).Older subjects had higher hsTnI values than younger subjects.

Example 7 Association of Myocardial Injury in TBI Patients and PoorOutcome

The objective of this study was to determine the association between TBIand serum high sensitivity troponin I (hsTnI) levels; identifyindependent risk factors for elevated hsTnI levels in TBI patients; anddetermine the association between hsTnI levels and functional outcomepost-TBI.

Analysis of the enrolled subjects, as described in Example 5, wasperformed. Emergency department (ED) patients who were 18 years or olderand were evaluated for either TBI or extra-cranial traumatic injury(Trauma Controls) were prospectively enrolled. Participants wereeligible for the TBI arm if they had blunt traumatic head injury,received a head CT scan as part of their ED evaluation, and met theAmerican College of Emergency Physicians' (ACEP) criteria for evaluationof TBI with a head CT scan. Participants in the trauma control arm weredeemed eligible if they were evaluated for a traumatic injury but didnot sustain head injury per self-report. Participants were excluded fromboth the TBI and trauma control arms if they met any of the followingcriteria: could not communicate in English, had no working telephonenumber, were pregnant, or had a past medical history of intracranialsurgery, intracranial hemorrhage, brain tumor or severe dementia.Patients transferred from other hospitals were eligible for enrollmentif they met study inclusion criteria and were enrolled within 24 hoursof injury.

Predictor Variables:

Data on patient demographics (age, gender, race, marital and employmentstatus), injury mechanism, and alcohol and recreational drug use within24 hours of injury were obtained directly from participants by trainedresearch coordinators who used structured data collection toolsrecommended as part of the National Institute of Neurological Disordersand Stroke (NINDS) Common Data Elements for TBI studies (Wilde et al.Arch Phys Med Rehabil. (2010) 91(11): 1650-1660). Data were entereddirectly into REDCap, an online electronic data entry and storage tool.Data on cardiovascular disease risk factors, which includedhypertension, high cholesterol, diabetes, coronary revascularization,congestive heart failure, estimated glomerular filtration rate (GFR) onthe day of injury, blood pressure, and heart rate at emergencydepartment presentation, were obtained by a structured review ofparticipant electronic medical records. Renal function was based on GFRand was categorized as normal, moderately reduced kidney function, andseverely reduced kidney function (>=60, 30-59, and <30 per mL/min/1.73m, respectively) (Zhang et al. J Stroke Cerebrovasc Dis. (2015) 24(10):2375-2384). Abbreviated Injury Scale (AIS) for the different bodyregions were coded by Digital Innovation Inc. using their proprietaryTri-Code software. AIS is a consensus derived, injury severity scoringsystem that classifies injuries by six body regions: (1)(Head/Neck/Cervical Spine; (2) Face (excluding frontal bone or skull);(3) Chest/Thoracic Spine; (4) Abdominal/Lumbar Spine/Pelvic Contents;(5) Extremities/Pelvic Girdle; and (6) Superficial/External includingBurns/Corrosions/Frostbite according to their relative severity on a 6point scale (1=minor and 6=unsurvivable) (Civil et al. J Trauma. (1988)28(1): 87-90). Since relatively few injuries were classified as serious,severe, critical or unsurvivable (AIS 3-6), all injuries with theseclassifications were reclassified as serious/more severe (AIS≥3).Patients without injury to a given body region were assigned a valueof 1. AIS determination was performed similarly for the TBI and traumacontrol groups.

Troponin Measurements.

500 TBI and 94 trauma control participants were studied. HsTnI wasmeasured in serum samples obtained at enrollment (initial blood draw)and at 4 hours after the initial blood draw in the TBI group and only atenrollment in the trauma control group. The initial blood sample wasobtained within 24 hours of injury and was aliquotted and stored in a−80 degree Celsius freezer within 2 hours of collection. HsTnI wasmeasured by the clinical chemistry lab at Johns Hopkins Hospital usingthe Abbott Architect STAT hsTnI assay. The limit of detection (LOD) ofthe assay was 2 ng/L. The gender-specific upper reference limit (URL)was 16 ng/L for females and 34 ng/L for males. The lowest hsTnIconcentration with a co-efficient of variation (CV) of 10% was 6 ng/L.The relative change in hsTnI at 4 hours in the TBI group was categorizedas: unchanged (between −20% and +20% difference in hsTnI levels betweeninitial blood draw and 4 hours after initial blood draw), decreasing(<−20% difference in hsTnI levels between initial blood draw and 4 hoursafter initial blood draw) or increasing (>+20% difference in hsTnIlevels between initial blood draw and 4 hours after initial blood draw).Elevated hsTnI was defined as hsTnI greater than the 99th %gender-neutral URL. Subjects with undetectable hsTnI were assigned avalue of 1 ng/L, which was the mid-point between 0 and the LOD.

Outcome Variables.

Head CT images were re-read by one board certified neuroradiologist. Thepresence/absence of traumatic lesions, including the size and locationof these lesions were classified according to the NINDS common dataelements for radiologic imaging of TBI (Duhaime et al. Arch Phys MedRehabil. (2010) 91(11): 1661-1666). Functional outcome was ascertainedusing the Extended Glasgow Outcome Scale (GOSE), which categorizesfunctional outcome on a scale of 1 (Dead) to 8 (upper good recovery).Severe disability/worse outcome was defined as GOSE<5 at 6 monthspost-injury.

Statistical Analysis.

Linear regression models were used to determine the differences ininitial troponin levels between the TBI and trauma control participantsand to identify independent predictors of hsTnI. These models wereadjusted for age and baseline head injury severity which were bothassociated with study group and hsTnI. To identify predictors of hsTnI,unadjusted and adjusted models were fit, the latter adjusting for knownpredictors of hsTnI that had statistically significant univariateassociations (p<0.05). These included: age, gender, race, hypertension,high cholesterol, diabetes, coronary revascularization, congestive heartfailure, renal function, blood pressure, and heart rate. The models werealso adjusted for injury severity as measured by abbreviated injuryseverity scores (AIS). To improve approximations to normality, hsTnIlevels were log-transformed prior to inclusion in the models. Hence alllinear regression parameters were interpreted as multiplicative on theexponential scale, and were reported as such. Unadjusted and adjustedlogistic regression models were also constructed to evaluate theassociations between initial hsTnI and the relative 4 hour change inhsTnI and functional recovery at 6 months. These models were adjustedfor age and head/neck/c-spine injury severity, both of which wereassociated with functional outcome post-TBI.

There was considerable missing data in the follow-up: 338 (68%) TBIparticipants had troponin measured at both time points and 337 (67%) hadfunctional outcome measured at 6 months. To consider the impact ofmissing data on the analysis, non-response weights equal to thereciprocal of the predicted probability of response at a given timepoint were constructed from a logistic regression of response onbaseline troponin, age, gender, race, marital status, employment status,and presence of an abnormal CT scan, and weighted generalized estimatingequation (GEE) analyses was conducted.

Statistical significance was defined at the α=0.05 level. Statisticalanalyses were conducting using SAS V 9.4 (SAS Institute, Cary, N.C.,2015).

Results:

Serum samples from 500 TBI and 94 trauma control participants wereassayed for hsTnI. The distribution of demographic characteristics andcardiovascular risk factors between TBI and trauma control participantswere similar with the exception of the TBI group being older (Table 21).TBI participants were more likely to have head and face injuries whereastrauma controls were more likely to have extremity, pelvic girdle andsuperficial/external injuries. Among TBI participants, 98 (19.6%) had atraumatic intracranial abnormality on head CT scan, and 339 (68%)completed follow-up assessment at 6 months post-injury. However, the6-month outcome data for two subjects could not be used because one hada stroke between 1 and 3 months and the other had repeat TBI between 3and 6 months. Among TBI participants with reliable follow-up data, 19(5.6%) had severe disability or worse.

hsTnI Values in Study Population.

The median time between injury and initial blood sampling was 4.4 hours(Interquartile range [IQR] 3.13-7.11) hours. Among TBI participants, 44(8.8%) had undetectable hsTnI, whereas among trauma controls, 16 (17.0%)had undetectable hsTnI. hsTnI values were higher within the TBI groupthan in trauma controls (median hsTnI: 5 [Interquartile range (IQR):3-11] ng/L versus 4 [IQR: 2-6] ng/L respectively), see FIG. 25. A hsTnIlevel cut-off of 5.7 ng/L at initial blood draw predicted a poor outcome(GOSE<5) at 1 month with a sensitivity of 83.3% and a specificity of54.9%. A hsTnI level cut-off of 5.6 ng/L at 4 hours blood draw predicteda poor outcome (GOSE<5) at 1 month with a sensitivity of 100% and aspecificity of 49.2%. A difference in hsTnI levels between initial blooddraw and 4 hours blood draw of 5.6 ng/L predicted a poor outcome(GOSE<5) at 1 month with a sensitivity of 82.4% and a specificity of69.5%.

Using the gender-specific cutoff for the URL, 52 (10.4%) participants inthe TBI group and 3 (3.2%) trauma controls had initial hsTnI valuesgreater than the 99th percentile (p=0.027). Initial hsTnI valuesremained higher in the TBI group after adjusting for age and head injuryseverity (regression co-efficient 1.66 [95% Confidence Interval (CI):1.29-2.12], p<0.001).

Unadjusted predictors of higher initial hsTnI levels within the TBIgroup included: older age, white race, history of hypertension, coronaryrevascularization, congestive heart failure, diabetes, high cholesterol,renal dysfunction, elevated mean arterial pressure at ED presentation,serious/more severe head/neck/c-spine injury, and serious/more severechest/t-spine injury. After adjusting for gender for confounders,independent predictors of higher initial hsTnI levels were: older age,male gender, history of congestive heart failure, history highcholesterol, elevated mean arterial pressure, renal dysfunction, andserious/more severe chest/T-spine injury (Table 22).

Among those with a 4 hour sample, 193 (57.1%), 62 (18.3%) and 83 (24.6%)had unchanged, decreasing and increasing hsTnI values, respectively.Initial hsTnI was associated with severe disability/worse outcome at 6months post-injury (odds ratio: 1.44 [95% CI: 1.02-2.02]). Thisassociation was no longer significant after adjusting for age andbaseline head injury severity (odds ratio: 1.04 (95% CI: 0.65-1.68)).Participants with increasing hsTnI at 4 hours had 4.52 (95% CI:1.56-13.11) times higher odds of having severe disability or worse at 6months post-injury than those with either unchanged or decreasing hsTnI,see FIG. 26. This association remained significant after adjusting forage and baseline head injury severity (odds ratio: 5.32 [95% CI:1.60-17.68]).

Imputation of Missing Data.

As a sensitivity analysis and alternative to the non-response weights,multiple imputation (MI) was used to impute the missing data in thehsTnI change variable and GOSE at 6 months-post injury. MI uses a modelto impute the missing data multiple times, using Rubin's combining rules(Schafer (1997) Analysis of incomplete multivariate data. Boca Raton:Chapman & Hall) to obtain inference about a parameter of interest (herethe regression coefficient associated with hsTnI changed over four hourson severe disability at 6 months). The point estimate was the mean ofthe imputed values and the variance estimate was a linear combination ofthe mean variance estimate using the fully imputed data and thebetween-imputation variance of the point estimate in each fully imputeddataset. To simultaneously impute both variables, a sequentialregression procedure (Raghunathan et al., Survey Methodology (2001) 27:85-95; Van Buuren (2007) Statistical Methods in Medical Research. 16:219-242) was employed in which the hsTnI change variable was imputedbased on the latest imputation of GOSE at 6 months-post injury, and thenthe GOSE at 6 months-post injury was imputed based on this imputation ofthe hsTnI change variable. Both imputations used multinomial logisticregression, and also conditioned on age, gender, cholesterol level,hypertension, diabetes, and presence of stent/CABG, baseline (log)hsTnI,and baseline injury status. This result was broadly consistent with thefinding using non-response weights, with participants with increasinghsTnI at 4 hours having 4.31 (95% CI: 1.55-11.93) times higher odds ofhaving severe disability or worse at 6 months post-injury than thosewith either unchanged or decreasing hsTnI, and 5.00 (95% CI: 1.36-18.42)times higher odds of having severe disability or worse at 6 monthspost-injury after adjusting for age and baseline head injury severity.The imputation of missing data did not result in a change in the overallconclusions.

TABLE 21 Demographic and clinical characteristics of study populationTrauma TBI Control p- n = 500 n = 94 value Median age in years (IQR) 44(28-63) 36 (26-56) 0.02 Female (%) 201 (40.2) 39 (41.5) 0.82 Race (%)0.58 White 266 (53.2) 52 (55.9) Black 198 (39.6) 37 (39.8) Other 36(7.2) 4 (4.3) Alcohol within 24 hours of 147 (29.4) 22 (23.4) 0.24enrollment Recreational drug within 24 62 (12.4) 7 (7.5) 0.28 hoursHypertension 178 (35.6) 27 (28.7) 0.20 Coronary revascularization 38(7.6) 2 (2.1) 0.05 Congestive heart failure 22 (4.4) 1 (1.1) 0.12Diabetes 65 (13.0) 8 (8.5) 0.22 High cholesterol 90 (18.0) 21 (22.3)0.32 Mean arterial pressure in 102 (93-114) 99 (99-110) 0.03 mmHg (IQR)Glomerular filtration rate 0.21 (mL/min/1.73 m²) ≥60 399 (87.5) 35(81.4) 30-59 51 (11.2) 6 (14.0) <30 6 (1.3) 2 (4.7) Mechanism <0.01 Fall176 (35.2) 31 (33.0) MVC 122 (24.4) 0 (0.0) Pedestrian Struck 49 (9.8) 1(1.1) Assault 87 (17.4) 0 (0.0) Cycle 39 (7.8) 0 (0.0) Struck by/against23 (4.6) 1 (1.1) Other 5 (0.8) 55 (58.5) AIS Severity: Head/Neck/ <0.01C-Spine 1 393 (78.6) 94 (100) 2 17 (3.4) 0 (0.0) 3 or greater 90 (18.0)0 (.0) AIS Severity: Face <0.01 1 452 (90.4) 94 (100.0) 2 48 (9.6) 0(0.0) 3 or greater 0 (0.0) 0 (0.0) AIS Severity: Chest/T-spine 0.19 1469 (93.8) 91 (96.8) 2 14 (2.8) 3 (3.2) 3 or greater 17 (3.4) 0 (0.0)AIS Severity: Abdomen/ 0.15 Pelvis/L-spine 1 489 (97.8) 94 (0.0) 2 11(2.2) 0 (0.0) 3 or greater 0 (0.0) 0 (0.0) AIS Severity: Extremities<0.01 and Pelvic girdle 1 467 (93.4) 44 (46.8) 2 28 (5.6) 43 (45.7) 3 orgreater 5 (1.0) 7 (7.5) AIS Severity: Superficial <0.01 and external 1467 (93.4) 44 (46.8) 2 28 (5.6) 43 (45.7) 3 or greater 5 (1.0) 17 3.4)

TABLE 22 Determinants of initial high sensitivity troponin I levelsUnivariable models Multivariable models* Regression Regressioncoefficient coefficient (95% confidence (95% confidence Characteristicinterval) P-value interval) P-value Age per decile 1.27 (1.21-1.33)<0.001 1.12 (1.04-1.20) 0.002 Female (%) 0.90 (0.73-1.11) 0.31 Race (%)White 1.00 (Reference) Black 0.73 (0.59-0.91) <0.001 1.07 (0.86-1.32)0.55 Other 0.47 (0.31-0.7) <0.001 0.71 (0.48-1.04) 0.08 Married (%) 1.14(0.91-1.43) 0.25 Employed (%) 1.40 (1.14-1.71) <0.001 Alcohol within 24hours 0.82 (0.65-1.02) 0.08 Recreational drug within 24 1.00 (0.98-1.01)0.79 hours Hypertension 1.97 (1.61-2.43) <0.001 0.87 (0.71-1.08) 0.21Coronary revascularization 3.26 (2.24-4.73) <0.001 0.99 (0.76-1.29) 0.94Congestive heart failure 3.77 (2.32-6.13) <0.001 1.21 (0.79-1.87) 0.38Diabetes 1.95 (1.45-2.63) <0.001 1.75 (1.05-2.92) 0.03 High cholesterol2.61 (2.02-3.36) <0.001 1.03 (0.75-1.41) 0.87 Mean arterial pressure per1.16 (1.10-1.24) <0.001 1.57 (1.14-2.16) <0.001 10 mmHg Heart rate per10 beats per 1.01 (0.96-1.07) 0.67 minute Glomerular filtration rate(mL/min/1.73 m²) ≥60 1.00 (Reference) 30-59 2.75 (2.03-3.71) <0.001 1.51(1.07-2.13) 0.02 <30 7.41 (3.45-15.9) <0.001 5.51 (2.31-13.12) <0.001Mechanism Fall 1.28 (0.87-1.87) 0.21 MVC 1.91 (1.33-2.75) <0.001Pedestrian Struck 1.44 (0.96-2.16) 0.08 Assault 1.04 (0.59-1.84) 0.89Cycle 0.96 (0.59-1.55) 0.86 Struck by/against 1.38 (0.43-4.46) 0.59Other 1.28 (0.87-1.87) 0.21 AIS Severity: Head/Neck/ C-Spine 1 1.00(Reference) 2 0.99 (0.56-1.74) 0.96 0.98 (0.58-1.65) 0.93 3 or greater1.33 (1.02-1.74) 0.04 1.11 (0.87-1.42) 0.40 AIS Severity: Face 1 1.00(Reference) 2 1.18 (0.83-1.66) 0.36 3 or greater NA NA AIS Severity:Chest/ T-spine 1 1.00 (Reference) 2 1.08 (0.58-2.01) 0.81 1.02(0.58-1.77) 0.95 3 or greater 1.98 (1.12-3.47) 0.02 1.75 (1.04-2.96)0.04 AIS Severity: Abdomen/Pelvis/L-spine 1 1.00 (Reference) 2 1.51(0.75-3.04) 0.25 3 or greater NA NA AIS Severity: Extremities and Pelvicgirdle 1 1.00 (Reference) 2 0.88 (0.56-1.37) 0.56 3 or greater 1.08(0.38-3.03) 0.88 AIS Severity: Superficial and external 1 1.00(Reference) 2 0.75 (0.24-2.38) 0.63 3 or greater NA NA

FIG. 27 shows AUC (0.7135) for the initial hsTnI. The initial hsTnIdiscriminates between subjects severe disability or worse at 1 monthversus those with better functional outcome (GOSE≥5) with an AUC of0.71. FIG. 28 shows the area under the receiver operator curve fordiscriminating between subjects with GOSE<5 versus those with GOSE≥5 at1 month using the 4 hour troponin. The AUC=0.77. FIG. 29 shows AUC thearea under the receiver operator curve for discriminating betweensubjects with 1 month GOSE<5 versus GOSE≥5 using the relative changebetween the initial and 4 hour troponin. FIG. 30 shows the area underthe receiver operator curve for discriminating between subjects with 1month GOSE<5 versus GOSE≥5 using the 24 hour hsTnI.

A logistic regression model was generated to incorporate age, hsTnIlevels at the initial time point, and hsTnI levels at a time point 4hours after the initial time point, as follows:

${\Pr\left( {Y = 1} \right)} = \frac{\exp\begin{pmatrix}{\beta_{0} + {\beta_{1}{\ln\left( {{initial}\mspace{14mu}{troponin}} \right)}} +} \\{{\beta_{2}{\ln\left( {4\mspace{14mu}{hour}\mspace{14mu}{troponin}} \right)}} + {\beta_{3}{age}}}\end{pmatrix}}{1 + {\exp\begin{pmatrix}{\beta_{0} + {\beta_{1}{\ln\left( {{initial}\mspace{14mu}{troponin}} \right)}} +} \\{{\beta_{2}{\ln\left( {4\mspace{14mu}{hour}\mspace{14mu}{troponin}} \right)}} + {\beta_{3}{age}}}\end{pmatrix}}}$The AUC of the model was 0.84 (see FIG. 31). Using this model, a cut-offvalue is a predicted probability of 0.036 and has a sensitivity of 94.1%and a specificity of 57.3% for identifying subjects with poor outcome(i.e., GOSE<5).

Myocardial injury occurred in TBI patients and it was independentlyassociated with poor functional outcome. See FIGS. 19-21 and 27-31.Elevated hsTnI values were found in TBI patients compared to traumacontrols. TBI subjects with increasing hsTnI were at risk for severedisability/worse outcome. HsTnI was detectable in the majority (91.2%)of patients evaluated for TBI and was elevated (>99th percentile) in10.4% of them. Furthermore, hsTnI values were higher in TBI patientscompared to those trauma controls. The present study provides additionalcorroborating evidence to support the occurrence of cardiac injury inTBI patients.

Participants with increasing hsTnI levels 4 hours after the initialsampling (on the day of injury) had a 5 times higher odds of havingsevere/worse disability at 6 months post-injury. Rising troponin levelsupon repeat measurement beyond values consistent with analyticalvariation, were indicative of acute processes. Patients with acutemyocardial injury, as signified by the increasing troponin values, maybe at risk for poor functional outcome post-TBI.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the disclosure, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the disclosure, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the disclosure are setout in the following numbered clauses:

Clause 1. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a suspected injury to the head to measure or detecta level of cardiac troponin I (cTnI); and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or a moderate to severe traumaticbrain injury when the level of cTnI in the sample is higher than areference level of cTnI or (2) a mild traumatic brain injury when thelevel of cTnI in the sample is lower than a reference level of cTnI.

Clause 2. The method of clause 1, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 3. The method of clause 2, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 4. The method of clause 3, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 5. The method of clause 4, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 6. The method of clause 2, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 7. The method of clause 6, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 8. The method of clause 7, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 9. The method of any one of clauses 1 to 8, wherein the referencelevel for cTnI is about 1.94 pg/mL, about 2.54 pg/mL, about 21.23 pg/mL,or about 43.79 pg/mL.

Clause 10. The method of any one of clauses 1 to 9, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 1 pg/mL to about 50 pg/mL.

Clause 11. The method of any one of clauses 1 to 10, wherein the sampleis taken within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the suspected injury to the head.

Clause 12. The method of any one of clauses 1 to 11, further comprisingtreating the subject assessed as having moderate to severe traumaticbrain injury with a traumatic brain injury treatment.

Clause 13. The method of any one of clauses 1 to 12, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 14. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained an injury to the head, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a suspected injury to the head to measure or detecta level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 15. The method of clause 14, wherein the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the suspected injury to the head.

Clause 16. The method of clause 14 or 15, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 17. The method of clause 16, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 18. The method of any one of clauses 14 to 17, wherein thereference level is correlated with positive head computed tomography.

Clause 19. The method of clause 18, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 20. The method of any one of clauses 14 to 19, wherein thereference level for cTnI is about 1.65 pg/mL, about 2.16 pg/mL, about14.75 pg/mL, or about 30.43 pg/mL.

Clause 21. The method of any one of clauses 14 to 20, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 1.0 pg/mL to about 50 pg/mL.

Clause 22. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 3 hours to about 6 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of moderate to severe traumatic braininjury if the level of troponin I detected has increased from the firstsample to the second sample and confirming the absence of mild traumaticbrain injury if the level of cTnI detected has remained unchanged or hasdecreased from the first sample to the second sample.

Clause 23. The method of clause 22, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of a suspected injury to the head.

Clause 24. The method of clause 22 or 23, wherein the subject has anabnormal head CT.

Clause 25. The method of any one of clauses 22 to 24, wherein the amountof cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 26. The method of any one of clauses 22 to 25, wherein the amountof cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 27. The method of any one of clauses 22 to 26, further comprisingtreating the subject assessed as having moderate to severe traumaticbrain injury with a traumatic brain injury treatment.

Clause 28. The method of any one of clauses 22 to 27, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 29. A method of aiding in the diagnosis and evaluation of a humansubject that has sustained or may have sustained an injury to the head,the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a suspected injury to the head to measure or detecta level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or a moderate to severe traumaticbrain injury when the level of cTnI in the sample is higher than areference level of cTnI or (2) a mild traumatic brain injury when thelevel of cTnI in the sample is lower than a reference level of cTnI.

Clause 30. The method of clause 29, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 31. The method of clause 30, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 32. The method of clause 31, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 33. The method of clause 32, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 34. The method of clause 30, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 35. The method of clause 34, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 36. The method of clause 35, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 37. The method of clause 36, wherein the reference level for cTnIis about 1.15 pg/mL or about 1.29 pg/mL.

Clause 38. The method of any one of clauses 29 to 37, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 0.5 pg/mL to about 30 pg/mL.

Clause 39. The method of any one of clauses 29 to 38, wherein the sampleis taken within about 5 minutes, within about 10 minutes, within about12 minutes, within about 15 minutes, within about 20 minutes, withinabout 30 minutes, within about 60 minutes, or within about 90 minutesafter a suspected injury to the head.

Clause 40. The method of any one of clauses 29 to 39, further comprisingtreating the subject assessed as having moderate to severe traumaticbrain injury with a traumatic brain injury treatment.

Clause 41. The method of any one of clauses 29 to 40, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 42. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained an injury to the head, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a suspected injury to the head to measure or detecta level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 43. The method of clause 42, wherein the subject has received aCT scan before or after the assay is performed.

Clause 44. The method of clause 43, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 45. The method of any one of clauses 42 to 44, wherein thereference level is correlated with positive head computed tomography.

Clause 46. The method of clause 45, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 47. The method of any one of clauses 42 to 46, wherein thereference level for cTnI is about 5.8 pg/mL or about 4.7 pg/mL.

Clause 48. The method of any one of clauses 42 to 47, wherein thereference level is

(a) determined by an assay having a sensitivity of between at leastabout 65% to 100% and a specificity of between at least about 30% to100%; (b) determined by an assay having a sensitivity of at least about85% and a specificity of at least about 33%; or (c) between at leastabout 0.5 pg/mL to about 25 pg/mL.

Clause 49. The method of any one of clauses 42 to 48, wherein the sampleis taken within about 5 minutes, within about 10 minutes, within about12 minutes, within about 15 minutes, within about 20 minutes, withinabout 30 minutes, within about 60 minutes, or within about 90 minutesafter a suspected injury to the head.

Clause 50. The method of any one of clauses 1 to 49, wherein measuringthe level of cTnI is done by an immunoassay or clinical chemistry assay.

Clause 51. The method of any one of clauses 1 to 50, wherein measuringthe level of cTnI comprises:

-   -   C. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex, such that a            cTnI-capture antibody-cTnI antigen-cTnI-detection antibody            complex is formed, and    -   D. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 52. The method of any one of clauses 1 to 51, wherein the sampleis selected from the group consisting of a whole blood sample, a serumsample, a cerebrospinal fluid sample, and a plasma sample.

Clause 53. The method of any one of clauses 1 to 52, wherein the sampleis obtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

Clause 54. The method of any one of clauses 1 to 53, wherein the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 55. The method of clause 54, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 56. The method of any one of clauses 1 to 53, wherein the sampleis obtained from a subject that suffers from an autoimmune disease, ametabolic disorder, a brain tumor, hypoxia, a virus, meningitis,hydrocephalus or combinations thereof.

Clause 58. The method of any one of clauses 1 to 56, wherein said methodcan be carried out on any subject without regard to factors selectedfrom the group consisting of the subject's clinical condition, thesubject's laboratory values, the subject's classification as sufferingfrom mild, moderate, severe, or a moderate to severe traumatic braininjury, the subject's exhibition of low or high levels of cTnI, and thetiming of any event wherein said subject may have sustained an injury tothe head.

Clause 59. The method of any one of clauses 1 to 56, wherein the sampleis a whole blood sample.

Clause 60. The method of any one of clauses 1 to 56, wherein the sampleis a serum sample.

Clause 61. The method of any one of clauses 1 to 56, wherein the sampleis a plasma sample.

Clause 62. The method of any one of clauses 58 to 60, wherein the assayis an immunoassay.

Clause 63. The method of any one of clauses 58 to 60, wherein the assayis a clinical chemistry assay.

Clause 64. The method of any one of clauses 58 to 60, wherein the assayis a single molecule detection assay.

Clause 65. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or suspected injury to the head tomeasure or detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI or (2) a mild traumatic braininjury when the level of cTnI in the sample is lower than a referencelevel of cTnI.

Clause 66. The method of clause 65, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 67. The method of clause 66, wherein the subject is suspected ashaving a moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 68. The method of clause 67, wherein the reference level iscorrelated with subjects having a moderate, severe, or moderate tosevere traumatic brain injury.

Clause 69. The method of clause 68, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 70. The method of clause 66, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 71. The method of clause 70, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 72. The method of clause 71, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 73. The method of any one of clauses 65 to 72, wherein thereference level for cTnI is about 1.94 pg/mL, about 2.54 pg/mL, about21.23 pg/mL, or about 43.79 pg/mL.

Clause 74. The method of any one of clauses 65 to 73, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 1 pg/mL to about 50 pg/mL.

Clause 75. The method of any one of clauses 65 to 74, wherein the sampleis taken within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 76. The method of any one of clauses 65 to 75, further comprisingtreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 77. The method of any one of clauses 65 to 76, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 78. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained a suspected injury to the head, themethod comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a suspected injury to the head to measure or detecta level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 79. The method of clause 78, wherein the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 80. The method of clause 78 or 79, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 81. The method of clause 80, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 82. The method of any one of clauses 78 to 81, wherein thereference level is correlated with positive head computed tomography.

Clause 83. The method of clause 82, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 84. The method of any one of clauses 78 to 83, wherein thereference level for cTnI is about 1.65 pg/mL, about 2.16 pg/mL, about14.75 pg/mL, or about 30.43 pg/mL.

Clause 85. The method of any one of clauses 78 to 84, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 1.0 pg/mL to about 50 pg/mL.

Clause 86. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 3 hours to about 6 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of a moderate, severe, or moderate tosevere traumatic brain injury if the level of cTnI detected hasincreased from the first sample to the second sample and confirming theabsence of mild traumatic brain injury if the level of cTnI detected hasremained unchanged or has decreased from the first sample to the secondsample.

Clause 87. The method of clause 86, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of a actual or suspected injury to thehead.

Clause 88. The method of clause 86 or 87, wherein the subject has anabnormal head CT.

Clause 89. The method of any one of clauses 86 to 88, wherein the amountof cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 90. The method of any one of clauses 86 to 89, wherein the amountof cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 91. The method of any one of clauses 86 to 90, further comprisingtreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 92. The method of any one of clauses 86 to 91, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 93. A method of aiding in the diagnosis and evaluation of a humansubject that has sustained or may have sustained an injury to the head,the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a actual or suspected injury to the head to measureor detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate,severe, or a moderate to severe traumatic brain injury (TBI), whereinthe subject is determined as having (1) a moderate, severe, or amoderate to severe traumatic brain injury when the level of cTnI in thesample is higher than a reference level of cTnI or (2) a mild traumaticbrain injury when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 94. The method of clause 93, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 95. The method of clause 94, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 96. The method of clause 95, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 97. The method of clause 96, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 98. The method of clause 97, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 99. The method of clause 98, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 100. The method of clause 99, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 101. The method of clause 100, wherein the reference level forcTnI is about 1.15 pg/mL or about 1.29 pg/mL.

Clause 102. The method of any one of clauses 93 to 101, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 0.5 pg/mL to about 30 pg/mL.

Clause 103. The method of any one of clauses 93 to 102, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 104. The method of any one of clauses 93 to 103, furthercomprising treating the subject assessed as having a moderate, severe,or moderate to severe traumatic brain injury with a traumatic braininjury treatment.

Clause 105. The method of any one of clauses 93 to 104, furthercomprising monitoring the subject assessed as having mild traumaticbrain injury.

Clause 106. A method of aiding in the determination of whether toperform a head computerized tomography (CT) scan on a human subject thathas sustained or may have sustained a suspected injury to the head, themethod comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a suspected injury to the head to measure or detecta level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 107. The method of clause 106, wherein the subject has received aCT scan before or after the assay is performed.

Clause 108. The method of clause 107, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 109. The method of any one of clauses 106 to 108, wherein thereference level is correlated with positive head computed tomography.

Clause 110. The method of clause 108, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 111. The method of any one of clauses 106 to 110, wherein thereference level for cTnI is about 5.8 pg/mL or about 4.7 pg/mL.

Clause 112. The method of any one of clauses 106 to 111, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 0.5 pg/mL to about 25 pg/mL.

Clause 113. The method of any one of clauses 106 to 112, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 114. The method of any one of clauses 65 to 113, whereinmeasuring the level of cTnI is done by an immunoassay or clinicalchemistry assay.

Clause 115. The method of any one of clauses 65 to 114, whereinmeasuring the level of cTnI comprises:

-   -   E. contacting the sample, either simultaneously or sequentially,        in any order with:    -   (1) a cTnI-capture antibody, which binds to an epitope on cTnI        or cTnI fragment to form a cTnI-capture antibody-cTnI antigen        complex, and    -   (2) a cTnI-detection antibody which includes a detectable label        and binds to an epitope on cTnI that is not bound by the        cTnI-capture antibody, to form a cTnI antigen-cTnI-detection        antibody complex, such that a cTnI-capture antibody-cTnI        antigen-cTnI-detection antibody complex is formed, and    -   F. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 116. The method of any one of clauses 65 to 115, wherein thesample is selected from the group consisting of a whole blood sample, aserum sample, a cerebrospinal fluid sample, and a plasma sample.

Clause 117. The method of any one of clauses 65 to 116, wherein thesample is obtained after the subject sustained an injury to the headcaused by physical shaking, blunt impact by an external mechanical orother force that results in a closed or open head trauma, one or morefalls, explosions or blasts or other types of blunt force trauma.

Clause 118. The method of any one of clauses 65 to 117, wherein thesample is obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 119. The method of clause 118, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 120. The method of any one of clauses 65 to 116, wherein thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

Clause 121. The method of any one of clauses 65 to 120, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or a moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of cTnI,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 122. The method of any one of clauses 65 to 121, wherein thesample is a whole blood sample.

Clause 123. The method of any one of clauses 65 to 121, wherein thesample is a serum sample.

Clause 124. The method of any one of clauses 65 to 121, wherein thesample is a plasma sample.

Clause 125. The method of any one of clauses 122 to 124, wherein theassay is an immunoassay.

Clause 126. The method of any one of clauses 122 to 124, wherein theassay is a clinical chemistry assay.

Clause 127. The method of any one of clauses 122 to 124, wherein theassay is a single molecule detection assay.

Clause 128. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a actual or suspected injury to the head to measureor detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI.

Clause 129. The method of clause 128, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 130. The method of clause 129, wherein the subject is suspectedas having moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 131. The method of clause 130, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 132. The method of clause 131, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 133. The method of clause 129, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 134. The method of clause 133, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 135. The method of clause 134, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 136. The method of any one of clauses 128 to 135, wherein thereference level for cTnI is about 1.94 pg/mL, about 2.54 pg/mL, about21.23 pg/mL, or about 43.79 pg/mL.

Clause 137. The method of any one of clauses 128 to 136, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 1 pg/mL to about 50 pg/mL.

Clause 138. The method of any one of clauses 128 to 137, wherein thesample is taken within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the actual or suspected injury to thehead.

Clause 139. A method of treating a mild or moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or suspected injury to the head tomeasure or detect a level of cTnI;

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 140. The method of clause 139, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 141. A method of aiding in the determination of whether toperform a head computerized tomography (CT) scan on a human subject thathas an actual or suspected injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a actual or suspected injury to the head to measureor detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 142. The method of clause 141, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 143. The method of clause 141 or 142, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 144. The method of clause 143, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 145. The method of any one of clauses 141 to 144, wherein thereference level is correlated with positive head computed tomography.

Clause 146. The method of clause 141, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 147. The method of any one of clauses 141 to 146, wherein thereference level for cTnI is about 1.65 pg/mL, about 2.16 pg/mL, about14.75 pg/mL, or about 30.43 pg/mL.

Clause 148. The method of any one of clauses 141 to 147, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 1.0 pg/mL to about 50 pg/mL.

Clause 149. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 3 hours to about 6 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of moderate, severe, or moderate to severetraumatic brain injury if the level of cTnI detected has increased fromthe first sample to the second sample and confirming the absence of mildtraumatic brain injury if the level of cTnI detected has remainedunchanged or has decreased from the first sample to the second sample.

Clause 150. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointand the second sample is taken from the human subject about 1 hours toabout 4 hours after the first sample, wherein the samples are biologicalsamples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of moderate to severe traumatic braininjury if the level of cTnI detected has increased from the first sampleto the second sample and confirming the absence of mild traumatic braininjury if the level of cTnI detected has remained unchanged or hasdecreased from the first sample to the second sample.

Clause 151. The method of clause 149 or 150, wherein the first sample istaken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of a actual or suspected injury to thehead.

Clause 152. The method of any of clauses 149 to 152, wherein the subjecthas an abnormal head CT.

Clause 153. The method of any one of clauses 149 to 152, wherein theamount of cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 154. The method of any one of clauses 149 to 153, wherein theamount of cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 155. A method of treating a mild or moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointand the second sample is taken from the human subject about 3 hours toabout 6 hours or about 1 hours to about 4 hours after the first sample,wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample;

c) confirming the occurrence of moderate to severe traumatic braininjury if the level of cTnI detected has increased from the first sampleto the second sample and confirming the absence of mild traumatic braininjury if the level of cTnI detected has remained unchanged or hasdecreased from the first sample to the second sample; and

d) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 156. The method of clause 155, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 157. A method of aiding in the diagnosis and evaluation of ahuman subject that has sustained or may have sustained an injury to thehead, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a actual or suspected injury to the head to measureor detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or a moderate, severe or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI or (2) a mild traumatic braininjury when the level of cTnI in the sample is lower than a referencelevel of cTnI.

Clause 158. The method of clause 157, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 159. The method of clause 158, wherein the subject is suspectedas having moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 160. The method of clause 159, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 161. The method of clause 160, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 162. The method of clause 157, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 163. The method of clause 158, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 164. The method of clause 163, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 165. The method of any one of clauses 157 to 164, wherein thereference level for cTnI is about 1.15 pg/mL or about 1.29 pg/mL.

Clause 166. The method of any one of clauses 157 to 165, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 0.5 pg/mL to about 30 pg/mL.

Clause 167. The method of any one of clauses 157 to 166, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 168. A method of treating a mild, moderate, severe, or moderateto severe TBI, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI;

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 169. The method of clause 168, further comprising monitoring thesubject assessed as having mild, moderate, severe, or moderate to severetraumatic brain injury.

Clause 170. A method of aiding in the determination of whether toperform a head computerized tomography (CT) scan on a human subject thathas an actual or suspected injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a actual or suspected injury to the head to measureor detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 171. The method of clause 170, wherein the subject has received aCT scan before or after the assay is performed.

Clause 172. The method of clause 171, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 173. The method of any one of clauses 170 to 172, wherein thereference level is correlated with positive head computed tomography.

Clause 174. The method of clause 170, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 175. The method of any one of clauses 170 to 174, wherein thereference level for cTnI is about 5.8 pg/mL or about 4.7 pg/mL.

Clause 176. The method of any one of clauses 170 to 175, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 0.5 pg/mL to about 25 pg/mL.

Clause 177. The method of any one of clauses 170 to 176, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 178. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin 24 hours after a injury to the head and the second sample istaken from the human subject about 0 to about 4 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI detected has increased from the first sample to the secondsample by at least about 20% and predicting a favorable outcome in thehuman subject if the level of cTnI detected has remained unchanged,decreased, or increased less than about 20% from the first sample to thesecond sample.

Clause 179. The method of clause 178, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the to the head.

Clause 180. The method of clause 178 or 179, wherein the method predictsthe outcome at about 1 month, 3 months, or 6 months after the injury tothe head.

Clause 181. The method of clause 180, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 182. The method of any one of clauses 178 to 181, wherein thesubject has a normal head CT scan.

Clause 183. The method of any one of clauses 178 to 182, wherein thesubject has received a Glasgow Coma Scale score before or after theassay is performed.

Clause 184. The method of clause any one of clauses 178 to 183, whereinthe subject has received a Glasgow Coma Scale score of 13-15 and issuspected as having mild traumatic brain injury based on the GlasgowComa Scale score.

Clause 185. The method of any one of clauses 178 to 184, wherein thesubject has received an Extended Glasgow Outcome Scale (GOSE) scorebefore or after the assay is performed.

Clause 186. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 28 hours after a injury to the head to measure or detect a levelof cTnI, wherein the sample is a biological sample; and

b) predicting an unfavorable outcome in the human subject if the levelof cTnI in the sample is higher than a reference level of cTnI andpredicting a favorable outcome in the human subject if the level of cTnIin the sample is lower than a reference level of cTnI.

Clause 187. The method of clause 186, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, withinabout 24 hours, within about 25 hours, within about 26 hours, withinabout 27 hours, or within about 28 hours of the injury to the head.

Clause 188. The method of clause 186 or 187, wherein the method predictsthe outcome at about 1 month, 3 months, or 6 months after the injury tothe head.

Clause 189. The method of clause 188, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 190. The method of any one of clauses 186 to 190, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 80% to 100% and a specificity of between at leastabout 45% to 100%; (b) determined by an assay having a sensitivity of atleast about 83.3% and a specificity of at least about 54.9%; (c)determined by an assay having a sensitivity of at least about 100% and aspecificity of at least about 49.2%; or (d) between at least about 1pg/mL to about 50 pg/mL.

Clause 191. The method of any one of clauses 186 to 190, wherein thereference level for cTnI is about 5.6 pg/mL or about 5.7 pg/mL.

Clause 192. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 4 hours after the first sample, wherein thesamples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI in the sample has increased from the first sample to the secondsample and predicting a favorable outcome in the human subject if thelevel of cTnI in the sample has remained unchanged or has decreased fromthe first sample to the second sample.

Clause 193. The method of clause 192, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours.

Clause 194. The method of clause 192 or 193, wherein the method predictsthe outcome at about 1 month, 3 months, or 6 months after the injury tothe head.

Clause 195. The method of clause 194, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 196. The method of any one of clauses 192 to 195, wherein theamount of cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 197. The method of any one of clauses 192 to 196, wherein theamount of cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 198. The method of any one of clauses 192 to 197, wherein thelevel of cTnI decreases or increase by at least an absolute amount fromthe first sample to the second sample.

Clause 199. The method of clause 198, wherein the absolute amount is (a)determined by an assay having a sensitivity of between at least about80% to 100% and a specificity of between at least about 45% to 100%; (b)determined by an assay having a sensitivity of at least about 82.4% anda specificity of at least about 69.5%; or (c) between at least about 1pg/mL to about 50 pg/mL.

Clause 200. The method of any one of clauses 199, wherein the absoluteamount is about 5.6 pg/mL.

Clause 201. The method of any one of clauses 192 to 200, furthercomprising treating the subject assessed as having an unfavorableoutcome with a traumatic brain injury treatment.

Clause 202. The method of any one of clauses 192 to 201, furthercomprising monitoring the subject assessed as having an unfavorableoutcome with a traumatic brain injury treatment.

Clause 203. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin 24 hours after a actual or suspected injury to the head and thesecond sample is taken from the human subject about 0 to about 4 hoursafter the first sample, wherein the samples are biological samples;

b) determining the age of the subject; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI in the first sample and/or second sample are higher than areference level of cTnI and the age of the subject is above a referenceage and predicting a favorable outcome in the human subject if the levelof cTnI in the first sample and second sample are lower than a referencelevels of cTnI and/or the age of the subject is below the reference age.

Clause 204. The method of any one clauses 128 to 203 further comprisingof treating the subject with at least one cardioprotective therapy.

Clause 205. The method of clause 204, wherein the at least onecardioprotective therapy comprises a beta-blocker, a diuretic, anAngiotensin-Converting Enzyme (ACE) inhibitor, a calcium channelblocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet,an anticlotting agent, an anticoagulation agent or combinations thereof.

Clause 206. The method of any one of clauses 128 to 205, whereinmeasuring the level of cTnI is done by an immunoassay or clinicalchemistry assay.

Clause 207. The method of any one of clauses 128 to 206, whereinmeasuring the level of cTnI comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex, such that a            cTnI-capture antibody-cTnI antigen-cTnI-detection antibody            complex is formed, and    -   B. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 208. The method of any one of clauses 128 to 207, wherein thesample is selected from the group consisting of a whole blood sample, aserum sample, a cerebrospinal fluid sample, and a plasma sample.

Clause 209. The method of any one of clauses 128 to 208, wherein thesample is obtained after the subject sustained an injury to the headcaused by physical shaking, blunt impact by an external mechanical orother force that results in a closed or open head trauma, one or morefalls, explosions or blasts or other types of blunt force trauma.

Clause 210. The method of any one of clauses 128 to 208, wherein thesample is obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 211. The method of clause 210, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 212. The method of any one of clauses 128 to 208, wherein thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

Clause 213. The method of any one of clauses 128 to 212, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or a moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of cTnI,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 214. The method of any one of clauses 128 to 214, wherein thesample is a whole blood sample.

Clause 215. The method of any one of clauses 128 to 214, wherein thesample is a serum sample.

Clause 216. The method of any one of clauses 128 to 214, wherein thesample is a plasma sample.

Clause 217. The method of any one of clauses 214 to 216, wherein theassay is an immunoassay.

Clause 218. The method of any one of clauses 214 to 216, wherein theassay is a clinical chemistry assay.

Clause 219. The method of any one of clauses 214 to 216, wherein theassay is a single molecule detection assay.

Clause 220. A method for evaluating a human subject for mild traumaticbrain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a actual or suspected injury to the head to measureor detect a level of cardiac troponin I (cTnI); and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI.

Clause 221. The method of clause 220, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 222. The method of clause 221, wherein the subject is suspectedas having moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 223. The method of clause 222, wherein the reference level iscorrelated with subjects having a moderate, severe or a moderate tosevere traumatic brain injury.

Clause 224. The method of clause 223, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 225. The method of clause 221, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 226. The method of clause 225, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 227. The method of clause 226, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 228. The method of any one of clauses 220 to 227, wherein thereference level for cTnI is about 1.94 pg/mL, about 2.54 pg/mL, about21.23 pg/mL, or about 43.79 pg/mL.

Clause 229. The method of any one of clauses 220 to 228, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 1 pg/mL to about 50 pg/mL.

Clause 230. The method of any one of clauses 220 to 228, wherein thesample is taken within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the actual or suspected injury to thehead.

Clause 231. A method of treating a mild or moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual actual or suspected injury to the head tomeasure or detect a level of cTnI;

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 232. The method of clause 12, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 233. A method of determining whether to perform a headcomputerized tomography (CT) scan on a human subject that has an actualor suspected injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after a actual or suspected injury to the head to measureor detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 234. The method of clause 233, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 235. The method of clauses 233 or 234, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 236. The method of clauses 235, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 237. The method of any one of clauses 233 to 236, wherein thereference level is correlated with positive head computed tomography.

Clause 238. The method of clauses 233, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 239. The method of any one of clauses 233 to 238, wherein thereference level for cTnI is about 1.65 pg/mL, about 2.16 pg/mL, about14.75 pg/mL, or about 30.43 pg/mL.

Clause 240. The method of any one of clauses 233 to 239, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 1.0 pg/mL to about 50 pg/mL.

Clause 241. A method for evaluating a human subject for mild traumaticbrain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 3 hours to about 6 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of moderate, severe, or moderate to severetraumatic brain injury if the level of cTnI detected has increased fromthe first sample to the second sample and confirming the absence of mildtraumatic brain injury if the level of cTnI detected has remainedunchanged or has decreased from the first sample to the second sample.

Clause 242. A method for evaluating a human subject for mild traumaticbrain injury in a human subject, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointand the second sample is taken from the human subject about 1 hours toabout 4 hours after the first sample, wherein the samples are biologicalsamples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) confirming the occurrence of moderate to severe traumatic braininjury if the level of cTnI detected has increased from the first sampleto the second sample and confirming the absence of mild traumatic braininjury if the level of cTnI detected has remained unchanged or hasdecreased from the first sample to the second sample.

Clause 243. The method of clauses 241 or 242, wherein the first sampleis taken from the subject within about 30 minutes, within about 1 hour,within about 2 hours, within about 3 hours, within about 4 hours, withinabout 5 hours, within about 6 hours, within about 7 hours, within about8 hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of a actual or suspected injury to thehead.

Clause 244. The method of any of clauses 241 to 243, wherein the subjecthas an abnormal head CT.

Clause 245. The method of any one of clauses 241 to 244, wherein theamount of cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 246. The method of any one of clauses 241 to 245, wherein theamount of cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 247. A method of treating a mild or moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointand the second sample is taken from the human subject about 3 hours toabout 6 hours or about 1 hours to about 4 hours after the first sample,wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample;

c) confirming the occurrence of moderate to severe traumatic braininjury if the level of cTnI detected has increased from the first sampleto the second sample and confirming the absence of mild traumatic braininjury if the level of cTnI detected has remained unchanged or hasdecreased from the first sample to the second sample; and

d) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 248. The method of clause 247, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 249. A method of aiding in the diagnosis and evaluation of ahuman subject that has sustained or may have sustained an injury to thehead, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after a actual or suspected injury to the head to measureor detect a level of cTnI; and

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI.

Clause 250. The method of clauses 249, wherein the subject has receiveda Glasgow Coma Scale score before or after the assay is performed.

Clause 251. The method of clause 250, wherein the subject is suspectedas having moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 252. The method of clause 251, wherein the reference level iscorrelated with subjects having a moderate, severe, or a moderate tosevere traumatic brain injury.

Clause 253. The method of clause 252, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 3-12.

Clause 254. The method of clause 250, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 255. The method of clause 254, wherein the reference level iscorrelated with subjects having mild traumatic brain injury.

Clause 256. The method of clause 255, wherein the reference level iscorrelated with a Glasgow Coma Scale score of 13-15.

Clause 257. The method of any one of clauses 249 to 256, wherein thereference level for cTnI is about 1.15 pg/mL or about 1.29 pg/mL.

Clause 258. The method of any one of clauses 249 to 257, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; or (c)between at least about 0.5 pg/mL to about 30 pg/mL.

Clause 259. The method of any one of clauses 249 to 258, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 260. A method of treating a mild, moderate, severe, or moderateto severe TBI, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI;

b) determining whether the subject has sustained a mild or a moderate tosevere traumatic brain injury (TBI), wherein the subject is determinedas having (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 261. The method of clauses 260, further comprising monitoring thesubject assessed as having mild, moderate, severe, or moderate to severetraumatic brain injury.

Clause 262. A method of aiding in the determination of whether toperform a head computerized tomography (CT) scan on a human subject thathas an actual or suspected injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and not performing a CTscan on the subject when the level of cTnI in the sample is lower than areference level of cTnI.

Clause 263. The method of clause 262, wherein the subject has received aCT scan before or after the assay is performed.

Clause 264. The method of clause 263, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 265. The method of any one of clauses 262 to 264, wherein thereference level is correlated with positive head computed tomography.

Clause 266. The method of clause 262, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 267. The method of any one of clauses 262 to 266, wherein thereference level for cTnI is about 5.8 pg/mL or about 4.7 pg/mL.

Clause 268. The method of any one of clauses 262 to 267, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 0.5 pg/mL to about 25 pg/mL.

Clause 269. The method of any one of clauses 262 to 268, wherein thesample is taken within about 5 minutes, within about 10 minutes, withinabout 12 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 60 minutes, or within about 90minutes after a actual or suspected injury to the head.

Clause 270. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin 24 hours after a injury to the head and the second sample istaken from the human subject about 0 to about 4 hours after the firstsample, wherein the samples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI detected has increased from the first sample to the secondsample by at least about 20% and predicting a favorable outcome in thehuman subject if the level of cTnI detected has remained unchanged,decreased, or increased less than about 20% from the first sample to thesecond sample.

Clause 271. The method of clauses 270, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the injury to the head.

Clause 272. The method of clauses 270 or 271, wherein the methodpredicts the outcome at about 1 month, 3 months, or 6 months after theactual or suspected injury to the head.

Clause 273. The method of clauses 272, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 274. The method of any one of clauses 270 to 273, wherein thesubject has a normal head CT scan.

Clause 275. The method of any one of clauses 270 to 274, wherein thesubject has received a Glasgow Coma Scale score before or after theassay is performed.

Clause 276. The method of claim any one of clauses 270 to 275, whereinthe subject has received a Glasgow Coma Scale score of 13-15 and issuspected as having mild traumatic brain injury based on the GlasgowComa Scale score.

Clause 277. The method of any one of clauses 270 to 276, wherein thesubject has received an Extended Glasgow Outcome Scale (GOSE) scorebefore or after the assay is performed.

Clause 278. A method for predicting the outcome of human subject havingmild traumatic brain injury, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 28 hours after a injury to measure or detect a level of cTnI,wherein the sample is a biological sample; and

b) predicting an unfavorable outcome in the human subject if the levelof cTnI in the sample is higher than a reference level of cTnI andpredicting a favorable outcome in the human subject if the level of cTnIin the sample is lower than a reference level of cTnI.

Clause 279. The method of claim 278, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, withinabout 24 hours, within about 25 hours, within about 26 hours, withinabout 27 hours, or within about 28 hours of the injury to the head.

Clause 280. The method of clauses 278 or 279, wherein the methodpredicts the outcome at about 1 month, 3 months, or 6 months after theinjury to the head.

Clause 281. The method of clause 280, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 282. The method of any one of clauses 278 to 281, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 80% to 100% and a specificity of between at leastabout 45% to 100%; (b) determined by an assay having a sensitivity of atleast about 83.3% and a specificity of at least about 54.9%; (c)determined by an assay having a sensitivity of at least about 100% and aspecificity of at least about 49.2%; or (d) between at least about 1pg/mL to about 50 pg/mL.

Clause 283. The method of any one of clauses 278 to 282, wherein thereference level for cTnI is about 5.6 pg/mL or about 5.7 pg/mL.

Clause 284. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin about 24 hours after head injury and the second sample is takenfrom the human subject about 4 hours after the first sample, wherein thesamples are biological samples;

b) determining whether the amount of cTnI has increased or decreasedfrom the first sample to the second sample; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI in the sample has increased from the first sample to the secondsample and predicting a favorable outcome in the human subject if thelevel of cTnI in the sample has remained unchanged or has decreased fromthe first sample to the second sample.

Clause 285. The method of clause 284, wherein the first sample is takenfrom the subject within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours.

Clause 286. The method of clauses 284 or 285, wherein the methodpredicts the outcome at about 1 month, 3 months, or 6 months after theinjury to the head.

Clause 287. The method of clause 286, wherein the subject is predictedto have an Extended Glasgow Outcome Scale (GOSE) score of 5 or less.

Clause 288. The method of any one of clauses 284 to 287, wherein theamount of cTnI in the first sample is from about 1.0 to about 50 pg/mL.

Clause 289. The method of any one of clauses 284 to 288, wherein theamount of cTnI in the second sample is from about 1.0 to about 50 pg/mL.

Clause 290. The method of any one of clauses 284 to 289, wherein thelevel of cTnI decreases or increase by at least an absolute amount fromthe first sample to the second sample.

Clause 291. The method of claim 290, wherein the absolute amount is (a)determined by an assay having a sensitivity of between at least about80% to 100% and a specificity of between at least about 45% to 100%; (b)determined by an assay having a sensitivity of at least about 82.4% anda specificity of at least about 69.5%; or (c) between at least about 1pg/mL to about 50 pg/mL.

Clause 292. The method of any one of clauses 291, wherein the absoluteamount is about 5.6 pg/mL.

Clause 293. The method of any one of clauses 270 to 292, furthercomprising treating the subject assessed as having an unfavorableoutcome with a traumatic brain injury treatment.

Clause 294. The method of any one of clauses 270 to 293, furthercomprising monitoring the subject assessed as having an unfavorableoutcome with a traumatic brain injury treatment.

Clause 295. A method for predicting the outcome of human subjects havingmild traumatic brain injury, the method comprising:

a) performing an assay on samples from the human subject to measure ordetect a level of cTnI in a first sample and a second sample, whereinthe first sample is taken from the human subject at a first time pointwithin 24 hours after a injury to the head and the second sample istaken from the human subject about 0 to about 4 hours after the firstsample, wherein the samples are biological samples;

b) determining the age of the subject; and

c) predicting an unfavorable outcome in the human subject if the levelof cTnI in the first sample and/or second sample are higher than areference level of cTnI and the age of the subject is above a referenceage and predicting a favorable outcome in the human subject if the levelof cTnI in the first sample and second sample are lower than a referencelevels of cTnI and/or the age of the subject is below the reference age.

Clause 296. The method of any one clauses 220 to 295 further comprisingtreating the subject with at least one cardioprotective therapy.

Clause 297. The method of clauses 296, wherein the at least onecardioprotective therapy comprises a beta-blocker, a diuretic, anAngiotensin-Converting Enzyme (ACE) inhibitor, a calcium channelblocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet,an anticlotting agent, an anticoagulation agent or combinations thereof.

Clause 298. The method of any one of clauses 220 to 297, whereinmeasuring the level of cTnI is done by an immunoassay or clinicalchemistry assay.

Clause 299. The method of any one of clauses 220 to 298, whereinmeasuring the level of cTnI comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex, such that a            cTnI-capture antibody-cTnI antigen-cTnI-detection antibody            complex is formed, and    -   B. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 300. The method of any one of clauses 220 to 299, wherein thesample is selected from the group consisting of a whole blood sample, aserum sample, a cerebrospinal fluid sample, and a plasma sample.

Clause 301. The method of any one of clauses 220 to 300, wherein thesample is obtained after the subject sustained an injury to the headcaused by physical shaking, blunt impact by an external mechanical orother force that results in a closed or open head trauma, one or morefalls, explosions or blasts or other types of blunt force trauma.

Clause 302. The method of any one of clauses 220 to 300, wherein thesample is obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 303. The method of clause 102, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 304. The method of any one of clauses 220 to 303, wherein thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

Clause 305. The method of any one of clauses 220 to 304, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or a moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of cTnI,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 306. The method of any one of clauses 220 to 305, wherein thesample is a whole blood sample.

Clause 307. The method of any one of clauses 220 to 305, wherein thesample is a serum sample.

Clause 308. The method of any one of clauses 220 to 305, wherein thesample is a plasma sample.

Clause 309. The method of any one of clauses 306 to 308, wherein theassay is an immunoassay.

Clause 310. The method of any one of clauses 306 to 308, wherein theassay is a clinical chemistry assay.

Clause 311. The method of any one of clauses 306 to 308, wherein theassay is a single molecule detection assay.

What is claimed is:
 1. A method comprising: a) performing at least oneassay for cardiac troponin I (cTnI) in at least one sample that is wholeblood, serum, plasma, or cerebrospinal fluid obtained from a subjectwithin about 24 hours after an actual or suspected injury to the head;and b) treating the subject for (1) a moderate, severe, or moderate tosevere traumatic brain injury (TBI) when the level of cTnI in the sampleis higher than a reference level of cTnI, or (2) a mild TBI when thelevel of cTnI in the sample is lower than a reference level of cTnI. 2.The method of claim 1, wherein the assay is selected from the groupconsisting of an immunoassay, a clinical chemistry assay, and apoint-of-care assay and/or the assay is performed using single moleculedetection.
 3. The method of claim 1, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed, andwherein the reference level is correlated with: (i) a Glasgow Coma Scalescore of 3-12 and the subject is suspected of having moderate, severe,or moderate to severe TBI based on the Glasgow Coma Scale score; or (ii)a Glasgow Coma Scale score of 13-15 and the subject is suspected ofhaving a mild TBI based on the Glasgow Coma Scale score.
 4. The methodof claim 1, wherein the reference level for cTnI is about 1.94 pg/mL,about 2.54 pg/mL, about 21.23 pg/mL, or about 43.79 pg/mL.
 5. The methodof claim 1, wherein the reference level is (a) determined by an assayhaving a sensitivity of between at least about 85% to 100% and aspecificity of between at least about 30% to 100%; (b) determined by anassay having a sensitivity of at least about 87.5% and a specificity ofat least about 31%; or (c) between at least about 1 pg/mL to about 50pg/mL.
 6. The method of claim 1, wherein the sample is taken withinabout 30 minutes, within about 1 hour, within about 2 hours, withinabout 3 hours, within about 4 hours, within about 5 hours, within about6 hours, within about 7 hours, within about 8 hours, within about 9hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of the actual or suspected injury to the head.
 7. The method ofclaim 1, further comprising monitoring the subject assessed as having amild, moderate, severe, or moderate to severe TBI.
 8. The method ofclaim 1, wherein the assay is performed on the sample obtained from thesubject with about 2 hours after an actual or suspected injury to thehead to measure or detect a level of cTnI.
 9. The method of claim 8,wherein the reference level for cTnI is about 1.15 pg/mL or about 1.29pg/mL.
 10. The method of claim 8, wherein the reference level is (a)determined by an assay having a sensitivity of between at least about85% to 100% and a specificity of between at least about 30% to 100%; (b)determined by an assay having a sensitivity of at least about 87.5% anda specificity of at least about 31%; or (c) between at least about 0.5pg/mL to about 30 pg/mL.
 11. The method of claim 8, wherein the sampleis taken within about 5 minutes, within about 10 minutes, within about12 minutes, within about 15 minutes, within about 20 minutes, withinabout 30 minutes, within about 60 minutes, or within about 90 minutesafter an actual or suspected injury to the head.
 12. A methodcomprising: a) performing at least one assay for cardiac troponin I(cTnI) in at least one sample that is whole blood, serum, plasma, orcerebrospinal fluid obtained from a subject within about 24 hours afteran actual or suspected injury to the head; and b) performing a headcomputed tomography (CT) scan on the subject when the level of cTnI inthe sample is higher than a reference level of cTnI.
 13. The method ofclaim 12, wherein the assay is selected from the group consisting of animmunoassay, a clinical chemistry assay, and a point-of-care assayand/or the assay is performed using single molecule detection.
 14. Themethod of claim 12, wherein the sample is taken from the subject withinabout 30 minutes, within about 1 hour, within about 2 hours, withinabout 3 hours, within about 4 hours, within about 5 hours, within about6 hours, within about 7 hours, within about 8 hours, within about 9hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of the actual or suspected injury to the head.
 15. The method ofclaim 12, wherein the reference level for cTnI is about 1.65 pg/mL,about 2.16 pg/mL, about 14.75 pg/mL, or about 30.43 pg/mL.
 16. Themethod of claim 12, wherein the reference level is (a) determined by anassay having a sensitivity of between at least about 65% to 100% and aspecificity of between at least about 30% to 100%; (b) determined by anassay having a sensitivity of at least about 85% and a specificity of atleast about 33%; or (c) between at least about 1.0 pg/mL to about 50pg/mL.
 17. The method of claim 12, wherein the assay is performed on thesample obtained from the subject with about 2 hours after an actual orsuspected injury to the head to measure or detect a level of cTnI. 18.The method of claim 17, wherein the reference level for cTnI is about5.8 pg/mL or about 4.7 pg/mL.
 19. The method of claim 17, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; or (c) betweenat least about 0.5 pg/mL to about 25 pg/mL.
 20. The method of claim 17,wherein the sample is taken within about 5 minutes, within about 10minutes, within about 12 minutes, within about 15 minutes, within about20 minutes, within about 30 minutes, within about 60 minutes, or withinabout 90 minutes after an actual or suspected injury to the head.
 21. Amethod comprising: a) performing at least one assay for cardiac troponinI (cTnI) in at least a first sample taken at a first time point and asecond sample taken at a second time point and obtained from a humansubject after an actual or suspected injury to the head; and b) treatingthe subject for mild, a moderate, severe, or moderate to severetraumatic brain injury (TBI) when the level of cTnI in the sample hasincreased from the first sample to the second sample; and wherein thesample is whole blood, serum, plasma, or cerebrospinal fluid obtainedwherein the first time point is within about 24 hours after the actualor suspected head injury and the second time point is within about 3 toabout 6 hours, and further wherein the subject levels of cTnI aredistinct from levels of cTnI in a control subject that has no TBI. 22.The method of claim 21, wherein the first sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.
 23. Themethod of claim 21, wherein the assay is selected from the groupconsisting of an immunoassay, a clinical chemistry assay, and apoint-of-care assay and/or the assay is performed using single moleculedetection.
 24. A method comprising: a) performing at least one assay forcardiac troponin I (cTnI) in at least a first sample taken at a firsttime point and a second sample taken at a second time point and obtainedfrom a human subject after an actual or suspected injury to the head;and b) treating the subject for mild, a moderate, severe, or moderate tosevere traumatic brain injury (TBI) when the level of cTnI in the samplehas increased from the first sample to the second sample; and whereinthe sample is whole blood, serum, plasma, or cerebrospinal fluidobtained wherein the first time point is within about 24 hours after theactual or suspected head injury and the second time point is withinabout 1 to about 4 hours, and further wherein the subject levels of cTnIare distinct from levels of cTnI in a control subject that has no TBI.25. The method of 24, wherein the first sample is taken from the subjectwithin about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of the actual or suspected injury to the head.
 26. The method ofclaim 24, wherein the assay is selected from the group consisting of animmunoassay, a clinical chemistry assay, and a point-of-care assayand/or the assay is performed using single molecule detection.
 27. Themethod of claim 24, wherein the amount of cTnI in the first sample orsecond sample is from about 1.0 to about 50 pg/mL.
 28. A methodcomprising: a) performing at least one assay for cardiac troponin I(cTnI) in at least a first sample taken at a first time point and asecond sample taken at a second time point and obtained from a humansubject after an actual or suspected injury to the head; and b) treatingthe subject for mild, a moderate, severe, or moderate to severetraumatic brain injury (TBI) when the level of cTnI in the sample hasincreased by at least about 20% from the first sample to the secondsample; and wherein the sample is whole blood, serum, plasma, orcerebrospinal fluid obtained wherein the first time point is withinabout 24 hours after the actual or suspected head injury and the secondtime point is within about 0 to about 4 hours, and further wherein thesubject levels of cTnI are distinct from levels of cTnI in a controlsubject that has no TBI.
 29. The method of claim 28, wherein the firstsample is taken from the subject within about 30 minutes, within about 1hour, within about 2 hours, within about 3 hours, within about 4 hours,within about 5 hours, within about 6 hours, within about 7 hours, withinabout 8 hours, within about 9 hours, within about 10 hours, within about11 hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the actual or suspected injury to thehead.
 30. The method of claim 28, wherein the assay is selected from thegroup consisting of an immunoassay, a clinical chemistry assay, and apoint-of-care assay and/or the assay is performed using single moleculedetection.